High-throughput profiling of drug interactions in Gram-positive bacteria

Antimicrobial resistance is an increasingly serious threat that influences all domains of modern medicine and public health. Many strategies are currently implemented to counteract its spread and tackle resistant strains. One of the most promising approaches is the use of drug combinations of approved compounds, bypassing the hurdles of new compound development.We tested more than 2000 drug combinations in different bacteria, including the species Staphylococcus aureus and Streptococcus pneumoniae, which are particularly relevant in the current antimicrobial resistance scenario. By probing several classes of antibiotics targeting different cellular processes, we showed how drug-drug interactions reflect the interplay between their targets, mirroring gene-gene interactions and capturing information on the mode of action of single drugs. Accordingly, drug interactions are subject to the same evolutionary constraints as cellular networks, with the conservation of drug interactions closely reflecting species phylogeny.We also investigated the impact of non-antibiotic drugs, which are commonly co-administered with antibiotics upon bacterial infections, testing these drugs in more than 3000 combinations with antibiotics in S. aureus. We highlighted both promising synergies, which were validated against multi-drug resistant S. aureus clinical isolates, and widespread antagonisms, which may hamper the efficacy of antimicrobial treatments.Finally, we integrated drug-drug and drug-gene interaction data, generated by testing single drugs on single-gene deletion mutant libraries. We were able to predict drug-drug interactions from drug-gene interactions using machine-learning approaches and uncover genes that are statistically associated with drug interactions, overall shedding light on the genetic background of drug interactions

Intermittent theta-burst stimulation rescues dopamine-dependent corticostriatal synaptic plasticity and motor behavior in experimental parkinsonism. Possible role of glial activity.

Background: Recent studies support the therapeutic utility of repetitive transcranial magnetic stimulation in Parkinson's disease (PD), whose progression is correlated with loss of corticostriatal long-term potentiation and long-term depression. Glial cell activation is also a feature of PD that is gaining increasing attention in the field because astrocytes play a role in chronic neuroinflammatory responses but are also able to manage dopamine (DA) levels. Methods: Intermittent theta-burst stimulation protocol was applied to study the effect of therapeutic neuromodulation on striatal DA levels measured by means of in vivo microdialysis in 6-hydroxydopamine-hemilesioned rats. Effects on corticostriatal synaptic plasticity were studied through in vitro intracellular and whole-cell patch clamp recordings while stepping test and CatWalk were used to test motor behavior. Immunohistochemical analyses were performed to analyze morphological changes in neurons and glial cells. Results: Acute theta-burst stimulation induced an increase in striatal DA levels in hemiparkinsonian rats, 80 minutes post-treatment, correlated with full recovery of plasticity and amelioration of motor performances. With the same timing, immediate early gene activation was restricted to striatal spiny neurons. Intense astrocytic and microglial responses were also significantly reduced 80 minutes following theta-burst stimulation. Conclusion: Taken together, these results provide a first glimpse on physiological adaptations that occur in the parkinsonian striatum following intermittent theta-burst stimulation and may help to disclose the real potential of this technique in treating PD and preventing DA replacement therapy-associated disturbances

Interazione tra anticorpi anti-peptidi citrullinati e sinoviociti nell'artrite reumatoide

L'artrite reumatoide, una delle più comuni malattie autoimmuni, è una malattia complessa ed eterogenea sia dal punto di vista eziopatogenetico che clinico, in cui fattori genetici e fattori ambientali concorrono ad una disregolazione della risposta immune sia innata che adattativa fino a determinare il danno articolare e le eventuali manifestazioni sistemiche. Le sue caratteristiche fondamentali sono la sinovite persistente, l'infiammazione sistemica e la presenza di autoanticorpi, in particolare il fattore reumatoide e gli anticorpi anti-peptidi citrullinati (ACPA). È progressivamente emerso come una delle popolazioni cellulari della sinovia, i sinoviociti di tipo fibroblastico (FLS), non sia meramente modificata dall'ambiente infiammatorio locale, ma partecipi attivamente alla distruzione della cartilagine, rivestendo un ruolo fondamentale nella patogenesi del danno articolare. In questo contesto, sembrano avere una funzione importante gli ACPA, che oltre ad essere marcatori diagnostici estremamente sensibili e specifici correlano con la prognosi della malattia ed hanno in ultima analisi permesso di individuare due sottotipi di AR, distinti per caratteristiche cliniche, genetiche e molecolari. Allo scopo di analizzare il ruolo degli ACPA nella produzione di citochine e chemochine da parte dei FLS, sono stati selezionati alcuni pazienti con AR nel cui siero fossero presenti ACPA a titolo elevato. Gli ACPA sono stati isolati dal siero mediante cromatografia di affinità su peptidi citrullinati immobilizzati su fase solida. La specificità degli anticorpi purificati è stata poi confermata mediante test ELISA. Da frammenti di tessuto sinoviale prelevato mediante sinoviectomie sono state approntate colture di FLS. Tutti gli esperimenti sono stati effettuati al terzo-quinto passaggio delle cellule in coltura. Gli ACPA sono stati incubati a tempi e concentrazioni differenti su sinoviociti prelevati da pazienti con AR oppure con osteoartrosi. Si è poi proceduto a valutare il pattern di citochine prodotte nel supernatante di coltura. In esperimenti preliminari è stato utilizzato un multiarray specifico per citochine umane ed è stato osservato un notevole aumento di produzione di CCL5/RANTES a seguito dell’incubazione dei FLS con APCA. Ulteriori conferme di questo risultato iniziale sono state ottenute mediante ELISA, basato su anticorpi monoclonali e policlonali specifici per CCL5/RANTES. Oltre al dosaggio della proteina nel supernatante, è stato dosato mediante RT-PCR anche l’mRNA codificante CCL5/RANTES ed è stato dimostrato un aumento dopo stimolo con ACPA. Al fine di analizzare le vie di signalling intracellulare coinvolte nella produzione di CCL5/RANTES, è stato effettuato uno screening mediante un sistema di tipo multiarray: nel caso dei sinoviociti stimolati con ACPA si ha una attivazione delle vie di fosforilazione di ERK1/2, p38, c-Jun, HSP27 e CREB maggiore rispetto a quanto rilevabile con le IgG di controllo. Per ottenere una quantificazione più precisa della misura in cui queste vie venissero attivate, sono state misurate mediante western blotting quantità relative delle forme fosforilate e non fosforilate di alcune delle proteine coinvolte, come ERK1/2, p38 e JNK. I dati ottenuti indicano che gli ACPA inducono produzione di CCL5/RANTES da parte dei FLS; tale produzione è particolarmente elevata quando i FLS derivano da pazienti con AR ed è regolata dall'attivazione dei componenti della cascata delle MAP chinasi, in particolare di ERK

Systematic analysis of drug combinations against Gram-positive bacteria

Drug combinations can expand options for antibacterial therapies but have not been systematically tested in Gram-positive species. We profiled ~8,000 combinations of 65 antibacterial drugs against the model species Bacillus subtilis and two prominent pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Thereby, we recapitulated previously known drug interactions, but also identified ten times more novel interactions in the pathogen S. aureus, including 150 synergies. We showed that two synergies were equally effective against multidrug-resistant S. aureus clinical isolates in vitro and in vivo. Interactions were largely species-specific and synergies were distinct from those of Gram-negative species, owing to cell surface and drug uptake differences. We also tested 2,728 combinations of 44 commonly prescribed non-antibiotic drugs with 62 drugs with antibacterial activity against S. aureus and identified numerous antagonisms that might compromise the efficacy of antimicrobial therapies. We identified even more synergies and showed that the anti-aggregant ticagrelor synergized with cationic antibiotics by modifying the surface charge of S. aureus. All data can be browsed in an interactive interface (https://apps.embl.de/combact/).</p

Systematic analysis of drug combinations against Gram-positive bacteria

Drug combinations can expand options for antibacterial therapies but have not been systematically tested in Gram-positive species. We profiled similar to 8,000 combinations of 65 antibacterial drugs against the model species Bacillus subtilis and two prominent pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Thereby, we recapitulated previously known drug interactions, but also identified ten times more novel interactions in the pathogen S. aureus, including 150 synergies. We showed that two synergies were equally effective against multidrug-resistant S. aureus clinical isolates in vitro and in vivo. Interactions were largely species-specific and synergies were distinct from those of Gram-negative species, owing to cell surface and drug uptake differences. We also tested 2,728 combinations of 44 commonly prescribed non-antibiotic drugs with 62 drugs with antibacterial activity against S. aureus and identified numerous antagonisms that might compromise the efficacy of antimicrobial therapies. We identified even more synergies and showed that the anti-aggregant ticagrelor synergized with cationic antibiotics by modifying the surface charge of S. aureus. All data can be browsed in an interactive interface (https://apps.embl.de/combact/).ISSN:2058-527

Understanding Factors Associated With Psychomotor Subtypes of Delirium in Older Inpatients With Dementia

Objectives: Few studies have analyzed factors associated with delirium subtypes. In this study, we investigate factors associated with subtypes of delirium only in patients with dementia to provide insights on the possible prevention and treatments. Design: This is a cross-sectional study nested in the \u201cDelirium Day\u201d study, a nationwide Italian point-prevalence study. Setting and Participants: Older patients admitted to 205 acute and 92 rehabilitation hospital wards. Measures: Delirium was evaluated with the 4-AT and the motor subtypes with the Delirium Motor Subtype Scale. Dementia was defined by the presence of a documented diagnosis in the medical records and/or prescription of acetylcholinesterase inhibitors or memantine prior to admission. Results: Of the 1057 patients with dementia, 35% had delirium, with 25.6% hyperactive, 33.1% hypoactive, 34.5% mixed, and 6.7% nonmotor subtype. There were higher odds of having venous catheters in the hypoactive (OR 1.82, 95% CI 1.18-2.81) and mixed type of delirium (OR 2.23, CI 1.43-3.46), whereas higher odds of urinary catheters in the hypoactive (OR 2.91, CI 1.92-4.39), hyperactive (OR 1.99, CI 1.23-3.21), and mixed types of delirium (OR 2.05, CI 1.36-3.07). We found higher odds of antipsychotics both in the hyperactive (OR 2.87, CI 1.81-4.54) and mixed subtype (OR 1.84, CI 1.24-2.75), whereas higher odds of antibiotics was present only in the mixed subtype (OR 1.91, CI 1.26-2.87). Conclusions and Implications: In patients with dementia, the mixed delirium subtype is the most prevalent followed by the hypoactive, hyperactive, and nonmotor subtype. Motor subtypes of delirium may be triggered by clinical factors, including the use of venous and urinary catheters, and the use of antipsychotics. Future studies are necessary to provide further insights on the possible pathophysiology of delirium in patients with dementia and to address the optimization of the management of potential risk factors