A New qPCR Assay for the Rapid Diagnosis of Anthonomus grandis Subspecies.

Rapid and accurate identification of Anthonomus grandis subspecies is crucial for effective management and eradication. Current diagnostic methods have limitations in terms of time to diagnosis (up to seven days) and can yield ambiguous results. Here, we present the validation of a custom TaqMan SNP Genotyping Assay for the rapid and accurate identification of A. grandis grandis (boll weevil) and A. g. thurberiae (thurberia weevil) subspecies. To validate the assay, we conducted three main experiments: (1) a sensitivity test to determine the DNA concentration range at which the assay performs, (2) a non-target specificity test to ensure no amplification in non-target weevils (false positives), and (3) an accuracy test comparing the results of the new assay to previously established methods. These experiments were carried out in parallel at three independent facilities to confirm the robustness of the assay to variations in equipment and personnel. We used DNA samples from various sources, including field-collected specimens, museum specimens, and previously isolated DNA. The assay demonstrated high sensitivity (PCR success with ≥0.05 ng/µL DNA template), specificity (0.02 false positive rate), and accuracy (97.7%) in diagnosing boll weevil and thurberia weevil subspecies. The entire workflow, including DNA extraction, assay preparation, PCR run time, and data analysis, can be completed within a single workday (7-9 h) by a single technician. The deployment of this assay as a diagnostic tool could benefit boll weevil management and eradication programs by enabling same-day diagnosis of trap-captured or intercepted weevil specimens. Furthermore, it offers a more reliable method for identifying unknown specimens, contributing to the overall effectiveness of boll weevil research and control efforts.

Exercise-based intervention as a nonsurgical treatment for patients with carpal instability: A case series.

BACKGROUND: Although the important roles of proprioception and neuromuscular control in carpal instabilities under laboratory conditions have been recognized, only a few studies have translated this knowledge into a routine clinical practice. PURPOSE: This study aimed to evaluate the results of a personalized rehabilitation in patients with carpal instability on functionality and pain intensity. STUDY DESIGN: This was a case series study. METHODS: This case series included 39 adults (mean age: 38.2 ± 14.0 years; 16/23 females/males) diagnosed with carpal instability (radial or ulnar) with indication for orthopedic treatment. The disabilities of the arm, shoulder, and hand questionnaire was used to assess upper limb functionality. Pain perception was assessed using a visual analog scale. Exercise-based physiotherapy interventions were performed according to the clinical needs of the patients for at least 6 weeks (2-3 sessions per week). For the treatment of radial instability (n = 13), strengthening exercises of the abductor pollicis longus, extensor carpi radialis longus, flexor carpi radialis, and pronator quadratus muscles were prescribed. For the treatment of ulnar instability (n = 24), extensor carpi ulnaris and pronator quadratus were trained. All patients underwent proprioceptive training in open kinetic chain and closed kinetic chain, as well as strengthening of the unaffected hand. Changes before and after treatment were compared using the nonparametric Wilcoxon signed rank test. RESULTS: A significant improvement with a large effect size in disabilities of the arm, shoulder, and hand (P < .001; d = 2.9) and visual analog scale (P < .001; d = 3.2) scores were obtained after treatment. Moreover, the changes were greater than the minimal clinically important difference of 10.8 and 1.4, respectively. Similar results were found when patients with radial instability and ulnar instability were analyzed separately. CONCLUSIONS: Personalized training with specific proprioception and strengthening exercises produces improvements in functionality and pain perception in our cohort of people with carpal instability. These results highlight the importance of multicomponent exercise in the treatment of wrist instability. Future randomized clinical trials should further investigate the effectiveness of this protocol.

A post-surgical rehabilitation program for women over 60 years old who underwent surgery in trauma and orthopedic hospital after distal radius fracture.

INTRODUCTION: Distal radius fractures (DRFs) are associated with a high economic burden and an impact on quality of life on these patients. Its high prevalence demonstrates the importance of registering functional results, with emphasis on vulnerable population such as elderly females. METHODS: Thirty-six patients were admitted to a multimodal rehabilitation program. Exercise progressions combined with manual therapy and electrophysical agents were provided, taking into account bone and soft tissues healing stages, protection of surgical intervention and symptoms of each patient. The primary outcome was the Patient Rated Wrist Evaluation (PRWE) questionnaire. The secondary outcomes were wrist and forearm range of motion (ROM), grip strength and lateral and tripod pinch strength. The paired t-test was used to compare mean PRWE, ROM, and strength between 6 and 12 weeks. RESULTS: An improvement of functionality was observed with a decreased of -20.9 (CI 95%: 25.9 to -15.9) points in the PRWE questionnaire (p < 0.001) between 6 and 12 weeks after DRF surgery. In addition, an increase in the ROM of the wrist (p < 0.001), grip strength (p < 0.001), lateral pinch (p < 0.001) and tripod pinch (p < 0.001) were observed between 6 and 12 weeks after surgery. CONCLUSIONS: These findings showed a clinically and statistically significant improvement in function, ROM, and strength assessed at weeks 6 and 12 after surgery. However, this study design cannot establish a cause-and-effect relationship. Future randomized controlled clinical trials should investigate the effectivity of similar rehabilitation programs.

Structure-Function Studies of Polymyxin B Lipononapeptides.

The emerging threat of infections caused by highly drug-resistant bacteria has prompted a resurgence in the use of the lipodecapeptide antibiotics polymyxin B and colistin as last resort therapies. Given the emergence of resistance to these drugs, there has also been a renewed interest in the development of next generation polymyxins with improved therapeutic indices and spectra of action. We report structure-activity studies of 36 polymyxin lipononapeptides structurally characterised by an exocyclic FA-Thr²-Dab³ lipodipeptide motif instead of the native FA-Dab¹-Thr²-Dab³ tripeptide motif found in polymyxin B, removing one of the positively charged residues believed to contribute to nephrotoxicity. The compounds were prepared by solid phase synthesis using an on-resin cyclisation approach, varying the fatty acid and the residues at position 2 (P2), P3 and P4, then assessing antimicrobial potency against a panel of Gram-negative bacteria, including polymyxin-resistant strains. Pairwise comparison of N-acyl nonapeptide and decapeptide analogues possessing different fatty acids demonstrated that antimicrobial potency is strongly influenced by the N-terminal L-Dab-1 residue, contingent upon the fatty acid. This study highlights that antimicrobial potency may be retained upon truncation of the N-terminal L-Dab-1 residue of the native exocyclic lipotripeptide motif found in polymyxin B. The strategy may aid in the design of next generation polymyxins.

Protein and Chemical Determinants of BL-1249 Action and Selectivity for K2P Channels.

K2P potassium channels generate leak currents that stabilize the resting membrane potential of excitable cells. Various K2P channels are implicated in pain, ischemia, depression, migraine, and anesthetic responses, making this family an attractive target for small molecule modulator development efforts. BL-1249, a compound from the fenamate class of nonsteroidal anti-inflammatory drugs is known to activate K2P2.1(TREK-1), the founding member of the thermo- and mechanosensitive TREK subfamily; however, its mechanism of action and effects on other K2P channels are not well-defined. Here, we demonstrate that BL-1249 extracellular application activates all TREK subfamily members but has no effect on other K2P subfamilies. Patch clamp experiments demonstrate that, similar to the diverse range of other chemical and physical TREK subfamily gating cues, BL-1249 stimulates the selectivity filter "C-type" gate that controls K2P function. BL-1249 displays selectivity among the TREK subfamily, activating K2P2.1(TREK-1) and K2P10.1(TREK-2) ∼10-fold more potently than K2P4.1(TRAAK). Investigation of mutants and K2P2.1(TREK-1)/K2P4.1(TRAAK) chimeras highlight the key roles of the C-terminal tail in BL-1249 action and identify the M2/M3 transmembrane helix interface as a key site of BL-1249 selectivity. Synthesis and characterization of a set of BL-1249 analogs demonstrates that both the tetrazole and opposing tetralin moieties are critical for function, whereas the conformational mobility between the two ring systems impacts selectivity. Together, our findings underscore the landscape of modes by which small molecules can affect K2P channels and provide crucial information for the development of better and more selective K2P modulators of the TREK subfamily.

Structure, Function, and Biosynthetic Origin of Octapeptin Antibiotics Active against Extensively Drug-Resistant Gram-Negative Bacteria.

Resistance to the last-resort antibiotic colistin is now widespread and new therapeutics are urgently required. We report the first in toto chemical synthesis and pre-clinical evaluation of octapeptins, a class of lipopeptides structurally related to colistin. The octapeptin biosynthetic cluster consisted of three non-ribosomal peptide synthetases (OctA, OctB, and OctC) that produced an amphiphilic antibiotic, octapeptin C4, which was shown to bind to and depolarize membranes. While active against multi-drug resistant (MDR) strains in vitro, octapeptin C4 displayed poor in vivo efficacy, most likely due to high plasma protein binding. Nuclear magnetic resonance solution structures, empirical structure-activity and structure-toxicity models were used to design synthetic octapeptins active against MDR and extensively drug-resistant (XDR) bacteria. The scaffold was then subtly altered to reduce plasma protein binding, while maintaining activity against MDR and XDR bacteria. In vivo efficacy was demonstrated in a murine bacteremia model with a colistin-resistant P. aeruginosa clinical isolate.

The Impact of Reduced Cardiac Rehabilitation on Maximal Treadmill Exercise Time: A RANDOMIZED CONTROLLED TRIAL.

PURPOSE: Cardiac rehabilitation programs (CRPs) remain underutilized partly because of access barriers. We therefore evaluated a CRP with fewer center-based sessions (rCRP) compared with standard CRP (sCRP) with respect to changes in exercise capacity and cardiac risk factors. METHODS: In this randomized controlled noninferiority trial, primary and secondary prevention patients at low and moderate risk were randomized to an sCRP (n = 60) or an rCRP (n = 61). Over 4 months, sCRP and rCRP participants attended 32 and 10 on-site cardiac rehabilitation sessions, respectively. The primary outcome was the difference in the change in exercise capacity from baseline at 4 and 16 months between the groups measured in seconds from a maximal treadmill exercise test. Noninferiority of the rCRP was tested with mixed-effects model analysis with a cut point of 60 seconds for the upper value of the group estimate. RESULTS: Attendance was higher for the rCRP group (97% ± 63% vs 71% ± 22%, P = .002). Over 16 months, exercise test time increased for the sCRP (524 ± 168 to 604 ± 172 seconds, P < .01) and the rCRP (565 ± 183 to 640 ± 192 seconds, P < .01). The rCRP was not inferior to the sCRP regarding changes in treadmill time (48.47 seconds, P = .454). The rCRP was not inferior to the sCRP regarding metabolic and anthropometric risk factors. CONCLUSION: Our findings suggest that, for a selected group of low-/moderate-risk patients, the number of center-based CRP exercise sessions can be decreased while maintaining reduced cardiovascular risk factors.

When is a medicine unwanted, how is it disposed, and how might safe disposal be promoted? Insights from the Australian population.

Objective The aim of the present study was to explore disposal practices of unwanted medicines in a representative sample of Australian adults, compare this with previous household waste surveys and explore awareness of the National Return and Disposal of Unwanted Medicines (RUM) Project. Methods A 10-min online survey was developed, piloted and conducted with an existing research panel of adult individuals. Survey questions recorded demographics, the presence of unwanted medicines in the home, medicine disposal practices and concerns about unwanted medicines. Descriptive statistical analyses and rank-ordered logit regression were conducted. Results Sixty per cent of 4302 respondents reported having unwanted medicines in their household. Medicines were primarily kept just in case they were needed again and one-third of these medicines were expired. Two-thirds of respondents disposed of medicines with the household garbage and approximately one-quarter poured medicines down the drain. Only 17.6% of respondents had heard of the RUM Project, although, once informed, 91.7% stated that they would use it. Respondents ranked the risk of unintended ingestion as the most important public health message for future social marketing campaigns. Conclusions Respondents were largely unaware of the RUM Project, yet were willing to use it once informed. Limited awareness could lead to environmental or public health risks, and targeted information campaigns are needed. What is known about the topic? There is a growing international evidence base on how people dispose of unwanted medicines and the negative consequences, particularly the environmental effects of inappropriate disposal. Although insight into variation in disposal methods is increasing, knowledge of how people perceive risks and awareness of inappropriate disposal methods is more limited. What does this paper add? This study provides evidence of inappropriate medicines disposal and potential stockpiling of medicines in Australian households that could contribute to environmental and/or public health risks. It also reveals possible trends towards a higher frequency of inappropriate disposal practices in the Australian context. Insights into respondents' perceptions of associated risks and awareness of a national scheme for appropriate disposal of medicines have not previously been reported. What are the implications for practitioners? The findings of the present study provide important insights for all health professionals as stakeholders in the quality use of medicines. It is important for those health professionals who assist consumers to manage their medicines to have strategies in place that routinely identify potential stockpiling and inform consumers about appropriate methods of medicines disposal. Although the findings of this study are specific to the Australian context, they may usefully inform policy, public health campaigns and the individual practices of health professionals and other stakeholders in promoting the quality use of medicines nationally and internationally.

Synthesis of octapeptin C4 and biological profiling against NDM-1 and polymyxin-resistant bacteria.

The first synthesis of octapeptin C4 was achieved using a combination of solid phase synthesis and off-resin cyclisation. Octapeptin C4 displayed antibiotic activity against multi-drug resistant, NDM-1 and polymyxin-resistant Gram-negative bacteria, with moderate activity against Staphylococcus aureus. The linear analogue of octapeptin C4 was also prepared, which showed reduced activity.

Cocientes urinarios Proteína-Creatinina y Albúmina-Creatinina en pacientes con lupus eritematoso sistémico / Urinary Protein/Creatinine ratio and Albumin/Creatinine ratio in patients with systemic lupus erythematosus / Quocientes urinários Proteína-Creatinina e Albumina- Creatinina em pacientes com lúpus eritematoso sistêmico

El objetivo del trabajo consistió en analizar las correlaciones entre: cociente Proteína/Creatinina en la primera orina de la mañana y Proteinuria de 24 horas (P/C-P24h); y cociente Albúmina/Creatinina en la primera orina de la mañana y Albuminuria de 24 horas (A/C-A24h) en pacientes con Lupus Eritematoso Sistémico y también evaluar la influencia del Clearance de Creatinina (ClCr) sobre la correlación P/C-P24h. Fue un estudio observacional, transversal y prospectivo. Se estudiaron 80 muestras de 52 pacientes lúpicos ambulatorios, entre marzo de 2013 y agosto de 2014. Se evaluaron mediante coeficiente de correlación de Spearman (CCS), las correlaciones P/C-P24h y A/C-A24h en distintos rangos de proteinuria y la influencia del ClCr sobre P/C-P24h. Para P/C-P24h cuando P24h<300 mg/24h CCS=0,6169 (n=52); cuando P24h≥300 mg/ 24h CCS=0,7461 (n=28). Para P/C-P24h en pacientes con ClCr<60 mL/ min CCS=0,9016 (n=12), y con ClCr>60 mL/min CCS=0,8689 (n=66). Para A/C-A24h a P24h<300 mg/24h CCS=0,8082 (n=37). Todos con p<0,0001. Este estudio mostró buena correlación P/C-P24h para P24h≥300 mg/24h y A/C-A24h para P24h<300 mg/24h. No se observó influencia del ClCr en la correlación P/C-P24h. Estos resultados sumados a los obtenidos por otros autores apoyan el uso del cociente A/C a P24h<300 mg/24h y P/C a P24h≥300 mg/24h para el seguimiento del compromiso renal en pacientes con Lupus Eritematoso Sistémico utilizando la primera orina de la mañana.

The objective of the present work was to analyze the correlation between: protein/creatinine ratio in first-morning urine and 24-hour urine protein (P/C-P24h), and albumin/creatinine ratio in first-morning urine and 24-hour urine albumin (A/C-A24h) in patients with Systemic Lupus Erythematosus, and to evaluate the influence of creatinine clearance (CrCl) on the P/C-P24h correlation.It was a prospective cross-sectional study in which 80 samples of 52 outpatients with lupus were studied between March 2013 and August 2014. They were evaluated by Spearman Correlation Coefficient (CCS), the P/C-P24h and A/C-A24h correlations in different ranges of proteinuria and the influence of ClCr on P/C-P24h.This study showed a good correlation P/C-P24h for P24h≥300 mg/24h and A/C-A24h for P24h<300 mg/24h. No influence of ClCr in the P/C-P24h correlation was observed. These results and those obtained by other authors support the use of the A/C to P24h<300 mg/24h ratio and P/C to P24h≥300 mg/24h ratio to monitor renal involvement in patients with systemic lupus erythematosus using the first-morning urine.

O objetivo do trabalho foi analisar as correlações entre quociente Proteína/Creatinina na primeira urina da manhã e Proteinúria de 24 horas (P/C-P24h); e quociente Albumina/Creatinina na primeira urina da manhã e Albuminuria de 24 horas (A/C-A24h) em paciêntes com Lupus Eritematoso Sistêmico e também avaliar a influência do Clearance de Creatinina (ClCr) sobre a correlação P/C-P24h. Foi um estudo observacional transversal e prospectivo. Foram estudadas 80 amostras de 52 pacientes ambulatórios com lúpus, entre março de 2013 e agosto de 2014. Avaliaram-se através do coeficiente de correlação de Spearman (CCS), as correlações P/C-P24h e A/C-A24h em diferentes níveis de proteinúria e a influência do ClCr sob P/C-P24h. Para P/C-P24h quando P24h<300 mg/24h CCS=0,6169 (n=52); quando P24h≥300 mg/24h CCS=0,7461 (n=28). Para P/C-P24h em pacientes com ClCr<60 mL/min CCS=0,9016 (n=12), e com ClCr>60mL/min CCS=0,8689 (n=66). Para A/C-A24h a P24h<300 mg/24h CCS=0,8082 (n=37). Em todos os casos, p<0,0001. Este estudo mostrou boa correlação P/C-P24h para P24h≥300 mg/24h y A/C-A24h para P24h<300 mg/24h. Não foi observada influência do ClCr na correlação P/C-P24h. Estes resultados, somados aos obtidos por outros autores, apoiam o uso do quociente A/C a P24h<300 mg/24h e P/C a P24h≥300 mg/24h para o seguimento do compromisso renal em pacientes com Lúpus Eritematoso Sistêmico utilizando a primeira urina da manhã.

Activity and Predicted Nephrotoxicity of Synthetic Antibiotics Based on Polymyxin B.

The polymyxin lipodecapeptides colistin and polymyxin B have become last resort therapies for infections caused by highly drug-resistant Gram-negative bacteria. Unfortunately, their utility is compromised by significant nephrotoxicity and polymyxin-resistant bacterial strains. We have conducted a systematic activity-toxicity investigation by varying eight of the nine polymyxin amino acid free side chains, preparing over 30 analogues using a novel solid-phase synthetic route. Compounds were tested against a panel of Gram-negative bacteria and counter-screened for in vitro cell toxicity. Promising compounds underwent additional testing against primary kidney cells isolated from human kidneys to better predict their nephrotoxic potential. Many of the new compounds possessed equal or better antimicrobial potency compared to polymyxin B, and some were less toxic than polymyxin B and colistin against mammalian HepG2 cells and human primary kidney cells. These initial structure-activity and structure-toxicity studies set the stage for further improvements to the polymyxin class of antibiotics.

Different 2-Aminothiazole Therapeutics Produce Distinct Patterns of Scrapie Prion Neuropathology in Mouse Brains.

Because no drug exists that halts or even slows any neurodegenerative disease, developing effective therapeutics for any prion disorder is urgent. We recently reported two compounds (IND24 and IND81) with the 2-aminothiazole (2-AMT) chemical scaffold that almost doubled the incubation times in scrapie prion-infected, wild-type (wt) FVB mice when given in a liquid diet. Remarkably, oral prophylactic treatment with IND24 beginning 14 days prior to intracerebral prion inoculation extended survival from ∼120 days to over 450 days. In addition to IND24, we evaluated the pharmacokinetics and efficacy of five additional 2-AMTs; one was not followed further because its brain penetration was poor. Of the remaining four new 2-AMTs, IND114338 doubled and IND125 tripled the incubation times of RML-inoculated wt and Tg4053 mice overexpressing wt mouse prion protein (PrP), respectively. Neuropathological examination of the brains from untreated controls showed a widespread deposition of self-propagating, ß-sheet-rich "scrapie" isoform (PrP(Sc)) prions accompanied by a profound astrocytic gliosis. In contrast, mice treated with 2-AMTs had lower levels of PrP(Sc) and associated astrocytic gliosis, with each compound resulting in a distinct pattern of deposition. Notably, IND125 prevented both PrP(Sc) accumulation and astrocytic gliosis in the cerebrum. Progressive central nervous system dysfunction in the IND125-treated mice was presumably due to the PrP(Sc) that accumulated in their brainstems. Disappointingly, none of the four new 2-AMTs prolonged the lives of mice expressing a chimeric human/mouse PrP transgene inoculated with Creutzfeldt-Jakob disease prions.

Lead identification to clinical candidate selection: drugs for Chagas disease.

Chagas disease affects 8 million people worldwide and remains a main cause of death due to heart failure in Latin America. The number of cases in the United States is now estimated to be 300,000, but there are currently no Food and Drug Administration (FDA)-approved drugs available for patients with Chagas disease. To fill this gap, we have established a public-private partnership between the University of California, San Francisco and the Genomics Institute of the Novartis Research Foundation (GNF) with the goal of delivering clinical candidates to treat Chagas disease. The discovery phase, based on the screening of more than 160,000 compounds from the GNF Academic Collaboration Library, led to the identification of new anti-Chagas scaffolds. Part of the screening campaign used and compared two screening methods, including a colorimetric-based assay using Trypanosoma cruzi expressing ß-galactosidase and an image-based, high-content screening (HCS) assay using the CA-I/72 strain of T. cruzi. Comparing molecules tested in both assays, we found that ergosterol biosynthesis inhibitors had greater potency in the colorimetric assay than in the HCS assay. Both assays were used to inform structure-activity relationships for antiparasitic efficacy and pharmacokinetics. A new anti-T. cruzi scaffold derived from xanthine was identified, and we describe its development as lead series.

Characterization of aerobically fit individuals with cardiovascular disease.

With an ageing population there is an increased prevalence of individuals living with cardiovascular disease (CVD). Characteristics of older aerobically fit individuals with previously diagnosed CVD have not been studied. Therefore, our knowledge is limited as to how, or if, aerobically fit individuals with CVD attempt to adapt their physical activity and the intensity of their training programmes. The objective of this paper is to characterise the physical activity habits and behaviours of older aerobically fit individuals with CVD. We identified 28 aerobically fit patients with CVD from those who completed a minimum of 15 and 12 min of the Bruce treadmill protocol for men and women, respectively. Consenting participants responded to questionnaires regarding physical activity levels, competitive event participation and self-monitoring since diagnosis of heart disease. Average age and treadmill time of participants were 56 and 49 years and 15.6 and 13.0 min for males and females, respectively. Data were obtained regarding recent medical history (medical diagnoses, surgeries/procedures). Despite the majority of individuals participating in the same or more activity since their diagnosis, 25% indicated that their condition limited their activity and 39% reported having symptoms during activity. Nearly all participants (93%) indicated that they monitored their heart rate during exercise. However, only 14% of participants stated that their physician advised them on how to exercise safely. It is necessary for physicians and cardiac rehabilitation programmes to be involved in safe and effective exercise programming to allow individuals to return to sport after CVD.

Antiprion compounds that reduce PrP(Sc) levels in dividing and stationary-phase cells.

During prion diseases, a normally benign, host protein, denoted PrP(C), undergoes alternative folding into the aberrant isoform, PrP(Sc). We used ELISA to identify and confirm hits in order to develop leads that reduce PrP(Sc) in prion-infected dividing and stationary-phase mouse neuroblastoma (ScN2a-cl3) cells. We tested 52,830 diverse small molecules in dividing cells and 49,430 in stationary-phase cells. This led to 3100 HTS and 970 single point confirmed (SPC) hits in dividing cells, 331 HTS and 55 confirmed SPC hits in stationary-phase cells as well as 36 confirmed SPC hits active in both. Fourteen chemical leads were identified from confirmed SPC hits in dividing cells and three in stationary-phase cells. From more than 682 compounds tested in concentration-effect relationships in dividing cells to determine potency (EC50), 102 had EC50 values between 1 and 10 µM and 50 had EC50 values of <1 µM; none affected cell viability. We observed an excellent correlation between EC50 values determined by ELISA and Western immunoblotting for 28 representative compounds in dividing cells (R(2)=0.75; p <0.0001). Of the 55 confirmed SPC hits in stationary-phase cells, 23 were piperazine, indole, or urea leads. The EC50 values of one indole in stationary-phase and dividing ScN2a-cl3 cells were 7.5 and 1.6 µM, respectively. Unexpectedly, the number of hits in stationary-phase cells was ~10% of that in dividing cells. The explanation for this difference remains to be determined.

A high-throughput functional screen identifies small molecule regulators of temperature- and mechano-sensitive K2P channels.

K2P (KCNK) potassium channels generate "leak" potassium currents that strongly influence cellular excitability and contribute to pain, somatosensation, anesthesia, and mood. Despite their physiological importance, K2Ps lack specific pharmacology. Addressing this issue has been complicated by the challenges that the leak nature of K2P currents poses for electrophysiology-based high-throughput screening strategies. Here, we present a yeast-based high-throughput screening assay that avoids this problem. Using a simple growth-based functional readout, we screened a library of 106,281 small molecules and identified two new inhibitors and three new activators of the mammalian K2P channel K2P2.1 (KCNK2, TREK-1). By combining biophysical, structure-activity, and mechanistic analysis, we developed a dihydroacridine analogue, ML67-33, that acts as a low micromolar, selective activator of temperature- and mechano-sensitive K2P channels. Biophysical studies show that ML67-33 reversibly increases channel currents by activating the extracellular selectivity filter-based C-type gate that forms the core gating apparatus on which a variety of diverse modulatory inputs converge. The new K2P modulators presented here, together with the yeast-based assay, should enable both mechanistic and physiological studies of K2P activity and facilitate the discovery and development of other K2P small molecule modulators.

2-Aminothiazoles with improved pharmacotherapeutic properties for treatment of prion disease.

Recently, we described the aminothiazole lead (4-biphenyl-4-ylthiazol-2-yl)-(6-methylpyridin-2-yl)-amine (1), which exhibits many desirable properties, including excellent stability in liver microsomes, oral bioavailability of ∼40 %, and high exposure in the brains of mice. Despite its good pharmacokinetic properties, compound 1 exhibited only modest potency in mouse neuroblastoma cells overexpressing the disease-causing prion protein PrP(Sc) . Accordingly, we sought to identify analogues of 1 with improved antiprion potency in ScN2a-cl3 cells while retaining similar or superior properties. Herein we report the discovery of improved lead compounds such as (6-methylpyridin-2-yl)-[4-(4-pyridin-3-yl-phenyl)thiazol-2-yl]amine and cyclopropanecarboxylic acid (4-biphenylthiazol-2-yl)amide, which exhibit brain exposure/EC50 ratios at least tenfold greater than that of compound 1.

Pharmacokinetics and metabolism of 2-aminothiazoles with antiprion activity in mice.

PURPOSE: To discover drugs lowering PrP(Sc) in prion-infected cultured neuronal cells that achieve high concentrations in brain to test in mouse models of prion disease and then treat people with these fatal diseases. METHODS: We tested 2-AMT analogs for EC50 and PK after a 40 mg/kg single dose and 40-210 mg/kg/day doses for 3 days. We calculated plasma and brain AUC, ratio of AUC/EC50 after dosing. We reasoned that compounds with high AUC/EC50 ratios should be good candidates going forward. RESULTS: We evaluated 27 2-AMTs in single-dose and 10 in 3-day PK studies, of which IND24 and IND81 were selected for testing in mouse models of prion disease. They had high concentrations in brain after oral dosing. Absolute bioavailability ranged from 27-40%. AUC/EC50 ratios after 3 days were >100 (total) and 48-113 (unbound). Stability in liver microsomes ranged from 30->60 min. Ring hydroxylated metabolites were observed in microsomes. Neither was a substrate for the MDR1 transporter. CONCLUSIONS: IND24 and IND81 are active in vitro and show high AUC/EC50 ratios (total and unbound) in plasma and brain. These will be evaluated in mouse models of prion disease.

Predictors of metabolic syndrome in participants of a cardiac rehabilitation program.

Metabolic syndrome increases the risk of all-cause mortality, cardiovascular mortality and cardiovascular events in patients with cardiovascular disease (CVD). This study assessed the predictors of metabolic syndrome, both its incidence and resolution in a cohort of cardiac rehabilitation program graduates. Methods. A total of 154 and 80 participants without and with metabolic syndrome respectively were followed for 48 months. Anthropometric measurements, metabolic risk factors, and quality of life were assessed at baseline and at 48 months. Logistic regression models were used to assess the predictors of metabolic syndrome onset and resolution. Results. Increasing waist circumference (OR 1.175, P ≤ 0.001) was an independent predictor for incident metabolic syndrome (R(2) for model = 0.46). Increasing waist circumference (OR 1.234, P ≤ 0.001), decreasing HDL-C (OR 0.027, P = 0.005), and increasing triglycerides (OR 3.005, P = 0.003) were predictors of metabolic syndrome resolution. Conclusion. Patients with CVD that further develop metabolic syndrome are particularly susceptible for the cascade of cardiovascular events and mortality. Increasing waist circumference confers a higher risk for future onset of metabolic syndrome in this group of patients. They will require closer follow-up and should be targeted for further prevention strategies after cardiac rehabilitation program completion.

Probing the steric space at the floor of the D1 dopamine receptor orthosteric binding domain: 7α-, 7ß-, 8α-, and 8ß-methyl substituted dihydrexidine analogues.

To probe the space at the floor of the orthosteric ligand binding site in the dopamine D(1) receptor, four methylated analogues of dihydrexidine (DHX) were synthesized with substitutions at the 7 and 8 positions. The 8α-axial, 8ß-equatorial, and 7α-equatorial were synthesized by photochemical cyclization of appropriately substituted N-benzoyl enamines, and the 7ß-axial analogue was prepared by an intramolecular Henry reaction. All of the methylated analogues displayed losses in affinity when compared to DHX (20 nM): 8ß-Me(ax)-DHX (270 nM), 8α-Me(eq)-DHX (920 nM), 7ß-Me(eq)-DHX (6540 nM), and 7α-Me(ax)-DHX (>10000 nM). Molecular modeling studies suggest that although the disruption of an aromatic interaction between Phe203(5.47) and Phe288(6.51) is the cause for the 14-fold loss in affinity associated with 8ß-axial substitution, unfavorable steric interactions with Ser107(3.36) result in the more dramatic decreases in binding affinity suffered by the rest of the analogues.