Investigation of the specificity and mechanism of action of the ULK1/AMPK inhibitor SBI-0206965

SBI-0206965, originally identified as an inhibitor of the autophagy initiator kinase ULK1, has recently been reported as a more potent and selective AMP-activated protein kinase (AMPK) inhibitor relative to the widely used, but promiscuous inhibitor Compound C/Dorsomorphin. Here, we studied the effects of SBI-0206965 on AMPK signalling and metabolic readouts in multiple cell types, including hepatocytes, skeletal muscle cells and adipocytes. We observed SBI-0206965 dose dependently attenuated AMPK activator (991)-stimulated ACC phosphorylation and inhibition of lipogenesis in hepatocytes. SBI-0206965 (≥25 μM) modestly inhibited AMPK signalling in C2C12 myotubes, but also inhibited insulin signalling, insulin-mediated/AMPK-independent glucose uptake, and AICA-riboside uptake. We performed an extended screen of SBI-0206965 against a panel of 140 human protein kinases in vitro, which showed SBI-0206965 inhibits several kinases, including members of AMPK-related kinases (NUAK1, MARK3/4), equally or more potently than AMPK or ULK1. This screen, together with molecular modelling, revealed that most SBI-0206965-sensitive kinases contain a large gatekeeper residue with a preference for methionine at this position. We observed that mutation of the gatekeeper methionine to a smaller side chain amino acid (threonine) rendered AMPK and ULK1 resistant to SBI-0206965 inhibition. These results demonstrate that although SBI-0206965 has utility for delineating AMPK or ULK1 signalling and cellular functions, the compound potently inhibits several other kinases and critical cellular functions such as glucose and nucleoside uptake. Our study demonstrates a role for the gatekeeper residue as a determinant of the inhibitor sensitivity and inhibitor-resistant mutant forms could be exploited as potential controls to probe specific cellular effects of SBI-0206965

In vitro biofilm formation by Staphylococcus aureus isolated from wounds of hospital-admitted patients and their association with antimicrobial resistance

Puja Neopane,1,2 Hari Prasad Nepal,3 Rojeet Shrestha,4 Osamu Uehara,5 Yoshihiro Abiko2 1Department of Microbiology, Chitwan Medical College and Teaching Hospital, Bharatpur, Nepal; 2Division of Oral Medicine and Pathology, Department of Human Biology and Pathophysiology, School of Dentistry, Health Sciences University of Hokkaido, Tobetsu, Japan; 3Department of Microbiology, Trinity School of Medicine, Kingstown, St. Vincent and the Grenadines; 4School of Medicine, Washington University of Barbados, St. Philip, Barbados; 5Division of Disease Control and Molecular Epidemiology, Department of Oral Growth and Development, Health Sciences University of Hokkaido, Tobetsu, Japan Introduction: Staphylococcus aureus including methicillin-resistant S. aureus (MRSA) has the propensity to form biofilms, and causes significant mortality and morbidity in the patients with wounds. Our aim was to study the in vitro biofilm-forming ability of S. aureus isolated from wounds of hospitalized patients and their association with antimicrobial resistance. Materials and methods: Forty-three clinical isolates of S. aureus were obtained from 150 pus samples using standard microbiological techniques. Biofilm formation in these isolates was detected by tissue culture plate (TCP) method and tube adherence method (TM). Antimicrobial susceptibility test was performed using the modified Kirby–Bauer disk diffusion method as per Clinical and Laboratory Standards Institute guidelines. MRSA was detected using the cefoxitin disk test.Results: Biofilm formation was observed in 30 (69.8%) and 28 (65.1%) isolates of S. aureus via TCP method and TM, respectively. Biofilm-producing S. aureus exhibited a higher incidence of antimicrobial resistance when compared with the biofilm nonproducers (P<0.05). Importantly, 86.7% of biofilm-producing S. aureus were multidrug resistant (MDR), whereas all the biofilm nonproducers were non-MDR (P<0.05). Large proportions (43.3%) of biofilm producers were identified as MRSA; however, none of the biofilm nonproducers were found to be MRSA (P<0.05). Conclusion: Both the in vitro methods showed that S. aureus isolated from wound infection of hospitalized patients have high degree of biofilm-forming ability. Biofilm-producing strains have very high tendency to exhibit antimicrobial resistance, multidrug resistance and methicillin resistance. Regular surveillance of biofilm formation by S. aureus and their antimicrobial resistance profile may lead to the early treatment of the wound infection. Keywords: biofilm, multidrug resistant, methicillin-resistant Staphylococcus aureu

Managing the sick child in the era of declining malaria transmission : development of ALMANACH, an electronic algorithm for appropriate use of antimicrobials

To review the available knowledge on epidemiology and diagnoses of acute infections in children aged 2 to 59 months in primary care setting and develop an electronic algorithm for the Integrated Management of Childhood Illness to reach optimal clinical outcome and rational use of medicines.; A structured literature review in Medline, Embase and the Cochrane Database of Systematic Review (CDRS) looked for available estimations of diseases prevalence in outpatients aged 2-59 months, and for available evidence on i) accuracy of clinical predictors, and ii) performance of point-of-care tests for targeted diseases. A new algorithm for the management of childhood illness (ALMANACH) was designed based on evidence retrieved and results of a study on etiologies of fever in Tanzanian children outpatients.; The major changes in ALMANACH compared to IMCI (2008 version) are the following: i) assessment of 10 danger signs, ii) classification of non-severe children into febrile and non-febrile illness, the latter receiving no antibiotics, iii) classification of pneumonia based on a respiratory rate threshold of 50 assessed twice for febrile children 12-59 months; iv) malaria rapid diagnostic test performed for all febrile children. In the absence of identified source of fever at the end of the assessment, v) urine dipstick performed for febrile children >2years to consider urinary tract infection, vi) classification of 'possible typhoid' for febrile children 5 year children and lead to more rational use of antimicrobials