-
Methods in Molecular Biology (Clifton,... 2024Current clinical practice assumes that a single antibiotic given as a bolus or as a course will successfully treat most infections. In modern medicine, this is becoming...
Current clinical practice assumes that a single antibiotic given as a bolus or as a course will successfully treat most infections. In modern medicine, this is becoming less and less true with drug-resistant, multi-drug-resistant, extensively drug-resistant, and untreatable infections becoming more common. Where single-drug therapy (monotherapy) fails, we will turn to multi-drug therapy. Alternatively, combination therapy could be useful to prevent the emergence of resistance. Multi-drug therapy is already standard for some multi-drug resistant infections and is the standard for the treatment of some pathogens such as Mycobacterium tuberculosis.The use of combination therapy for everyday infections could be a clear course out of the current AMR crisis we are facing. With every additional drug added to a combination (n + 1) the likelihood of the pathogen evolving resistance drops exponentially.Many generic antibiotics are cheap to manufacture as they have fallen out of patent protection but are less effective at pharmacologically effective doses due to overuse in the past. Combination therapy can combine these generic compounds into cocktails that can not only treat susceptible and resistant infections but can also reduce the risk of new resistances arising and can resuscitate the use of antimicrobials once thought defunct.In this chapter, we will summarize theory behind combination therapy and standard in vitro methodologies used.
Topics: Humans; Drug Therapy, Combination; Anti-Bacterial Agents; Microbial Sensitivity Tests; Anti-Infective Agents; Drug Resistance, Multiple, Bacterial
PubMed: 38949699
DOI: 10.1007/978-1-0716-3981-8_5 -
Methods in Molecular Biology (Clifton,... 2024Antimicrobial resistance (AMR) poses a serious threat to global health, potentially causing 10 million deaths per year globally by 2050. To tackle AMR, researchers from...
Antimicrobial resistance (AMR) poses a serious threat to global health, potentially causing 10 million deaths per year globally by 2050. To tackle AMR, researchers from all around the world have generated a selection of various formulated (viz. nanoparticulate, liposomal) therapeutic combinations to be evaluated for new antimicrobial drug discovery. To meet the urgent need for accelerating new antibacterial drug development, we need rapid but reliable whole-cell assay methods and models to test formulated therapeutic combinations against several pathogens in different in vitro conditions as models of actual infections.Over the past two decades, high-throughput spot-culture growth inhibition assay (HT-SPOTi) has been demonstrated to be a gold-standard drug susceptibility method for evaluating novel chemotherapeutic entities and existing drugs against various microbes of global concern. Our modified HT-SPOTi method serves the purpose of evaluating drug combinations against Gram-positive/negative microorganisms as well as acid-fast bacilli. The newly developed and modified HT-SPOTi assay builds upon the limitations of our previously published method to incorporate antimicrobial susceptibility testing with formulated therapeutic combinations. The modified HT-SPOTi is compared with a range of other antimicrobial susceptibility testing methods and validated using a library of existing antibiotics as well as formulated therapeutic combinations. The modified HT-SPOTi assay can serve as an efficient and reliable high-throughput drug screening platform to discover new potential antimicrobial molecules, including as part of therapeutic formulations.This chapter describes the generation of drug susceptibility profile for formulated therapeutic combinations using modified HT-SPOTi in a semi-automated system.
Topics: Microbial Sensitivity Tests; Anti-Bacterial Agents; High-Throughput Screening Assays; Humans; Bacteria
PubMed: 38949698
DOI: 10.1007/978-1-0716-3981-8_4 -
Methods in Molecular Biology (Clifton,... 2024Mycobacterium tuberculosis is the main causative agent of tuberculosis (TB)-an ancient yet widespread global infectious disease to which 1.6 million people lost their...
Mycobacterium tuberculosis is the main causative agent of tuberculosis (TB)-an ancient yet widespread global infectious disease to which 1.6 million people lost their lives in 2021. Antimicrobial resistance (AMR) has been an ongoing crisis for decades; 4.95 million deaths were associated with antibiotic resistance in 2019. While AMR is a multi-faceted problem, drug discovery is an urgent part of the solution and is at the forefront of modern research.The landscape of drug discovery for TB has undoubtedly been transformed by the development of high-throughput gene-silencing techniques that enable interrogation of every gene in the genome, and their relative contribution to fitness, virulence, and AMR. A recent advance in this area is CRISPR interference (CRISPRi). The application of this technique to antimicrobial susceptibility testing (AST) is the subject of ongoing research in basic science.CRISPRi technology can be used in conjunction with the high-throughput SPOT-culture growth inhibition assay (HT-SPOTi) to rapidly evaluate and assess gene essentiality including non-essential, conditionally essential (by using appropriate culture conditions), and essential genes. In addition, the HT-SPOTi method can develop drug susceptibility and drug resistance profiles.This technology is further useful for drug discovery groups who have designed target-based inhibitors rationally and wish to validate the primary mechanisms of their novel compounds' antibiotic action against the proposed target.
Topics: Microbial Sensitivity Tests; Mycobacterium tuberculosis; Drug Discovery; Gene Silencing; Humans; CRISPR-Cas Systems; Antitubercular Agents; Anti-Bacterial Agents; High-Throughput Screening Assays; Drug Resistance, Bacterial; Tuberculosis
PubMed: 38949697
DOI: 10.1007/978-1-0716-3981-8_3 -
Methods in Molecular Biology (Clifton,... 2024There is an increasing need for new treatment regimens to combat antibiotic-resistant strains of bacteria. Staphylococcus aureus is a clinically important, opportunist...
There is an increasing need for new treatment regimens to combat antibiotic-resistant strains of bacteria. Staphylococcus aureus is a clinically important, opportunist pathogen that has developed resistance to a range of antibiotics. The zebrafish larval model of systemic disease has been increasingly utilized to elucidate S. aureus virulence mechanisms and host-pathogen interactions. Here, we outline how this model can be used to investigate the effects of different antibiotics alone and in combination against S. aureus.
Topics: Animals; Zebrafish; Anti-Bacterial Agents; Staphylococcus aureus; Larva; Staphylococcal Infections; Disease Models, Animal; Drug Therapy, Combination; Host-Pathogen Interactions; Microbial Sensitivity Tests
PubMed: 38949695
DOI: 10.1007/978-1-0716-3981-8_1 -
Journal of Neuroimmune Pharmacology :... Jul 2024Amorfrutin B is a selective PPARγ modulator that we demonstrated to be a promising neuroprotective compound in cellular models of stroke and perinatal asphyxia....
Amorfrutin B Compromises Hypoxia/Ischemia-induced Activation of Human Microglia in a PPARγ-dependent Manner: Effects on Inflammation, Proliferation Potential, and Mitochondrial Status.
Amorfrutin B is a selective PPARγ modulator that we demonstrated to be a promising neuroprotective compound in cellular models of stroke and perinatal asphyxia. Although neuronal mechanisms of amorfrutin B-evoked neuroprotection have been identified, none of them reflects the actions of the compound on microglia, which play a pivotal role in brain response to hypoxia/ischemia. Here, we provide evidence for amorfrutin B-induced effects on human microglia subjected to hypoxia/ischemia; the compound counteracts inflammation, and influences mitochondrial status and proliferation potential in a PPARγ-dependent manner. Post-treatment with amorfrutin B decreased the IBA1 fluorescence intensity, reduced caspase-1 activity, and downregulated IL1B/IL-1β and TNFA but not IL10/IL-10 expression, which was upregulated. Amorfrutin B also stimulated PPARγ signaling, as evidenced by increased mRNA and/or protein levels of PPARγ and PGC1α. In addition, amorfrutin B reversed the hypoxia/ischemia-evoked effects on mitochondria-related parameters, such as mitochondrial membrane potential, BCL2/BCL2 expression and metabolic activity, which were correlated with diminished proliferation potential of microglia. Interestingly, the inhibitory effect of amorfrutin B on the proliferation potential and mitochondrial function of microglia is opposite to the stimulatory effect of amorfrutin B on mouse neuronal survival, as evidenced by increased neuronal viability and reduced neurodegeneration. In summary, this study showed for the first time that amorfrutin B compromises hypoxia/ischemia-induced activation of human microglia in a PPARγ-dependent manner, which involves inhibiting inflammation, normalizing mitochondrial status, and controlling proliferation potential. These data extend the protective potential of amorfrutin B in the pharmacotherapy of hypoxic/ischemic brain injury, targeting not only neurons but also activated microglia.
Topics: PPAR gamma; Humans; Microglia; Cell Proliferation; Mitochondria; Hypoxia-Ischemia, Brain; Inflammation; Cells, Cultured; Neuroprotective Agents
PubMed: 38949694
DOI: 10.1007/s11481-024-10135-9 -
ELife Jul 2024Secreted chemokines form concentration gradients in target tissues to control migratory directions and patterns of immune cells in response to inflammatory stimulation;...
Secreted chemokines form concentration gradients in target tissues to control migratory directions and patterns of immune cells in response to inflammatory stimulation; however, how the gradients are formed is much debated. Heparan sulfate (HS) binds to chemokines and modulates their activities. In this study, we investigated the roles of HS in the gradient formation and chemoattractant activity of CCL5 that is known to bind to HS. CCL5 and heparin underwent liquid-liquid phase separation and formed gradient, which was confirmed using CCL5 immobilized on heparin-beads. The biological implication of HS in CCL5 gradient formation was established in CHO-K1 (wild-type) and CHO-677 (lacking HS) cells by Transwell assay. The effect of HS on CCL5 chemoattractant activity was further proved by Transwell assay of human peripheral blood cells. Finally, peritoneal injection of the chemokines into mice showed reduced recruitment of inflammatory cells either by mutant CCL5 (lacking heparin-binding sequence) or by addition of heparin to wild-type CCL5. Our experimental data propose that co-phase separation of CCL5 with HS establishes a specific chemokine concentration gradient to trigger directional cell migration. The results warrant further investigation on other heparin-binding chemokines and allows for a more elaborate insight into disease process and new treatment strategies.
Topics: Chemokine CCL5; Animals; Heparitin Sulfate; Humans; Cricetulus; CHO Cells; Mice; Chemotaxis; Heparin; Phase Separation
PubMed: 38949655
DOI: 10.7554/eLife.93871 -
Natural Product Research Jul 2024This study aimed to investigate the potential anti-inflammatory properties of aqueous extract of (AEMV) using various animal models. Several inflammatory models...
This study aimed to investigate the potential anti-inflammatory properties of aqueous extract of (AEMV) using various animal models. Several inflammatory models including xylene-induced ear edoema, carrageenan-induced paw edoema, and Freund's adjuvant-induced arthritis were employed to evaluate the anti-inflammatory effects of AEMV. LC-MS/MS of AEMV revealed that the major component was Marrubiin, a diterpenoid lactone. AEMV demonstrated significant anti-inflammatory effects in all animal models tested. It effectively reduced ear and paw edoema induced by xylene and carrageenan, respectively. Furthermore, AEMV attenuated arthritis symptoms and hyperalgesia in rats with Freund's adjuvant-induced arthritis. Biochemical analyzes revealed normalisation of inflammatory markers, including C-reactive protein (CRP) levels, in treated animals. The findings suggest that AEMV possesses promising anti-inflammatory properties, supporting its potential therapeutic application in inflammatory conditions such as arthritis. Further investigations are needed to clarify the underlying mechanisms and optimise dosing regimens for clinical use.
PubMed: 38949651
DOI: 10.1080/14786419.2024.2372656 -
Translational Vision Science &... Jul 2024We sought to evaluate the efficacy of growth differentiation factor (GDF)-15 treatment for suppressing epithelial-mesenchymal transition (EMT) and alleviating...
PURPOSE
We sought to evaluate the efficacy of growth differentiation factor (GDF)-15 treatment for suppressing epithelial-mesenchymal transition (EMT) and alleviating transforming growth factor β2 (TGFβ2)-induced lens opacity.
METHODS
To test whether GDF-15 is a molecule that prevents EMT, we pretreated the culture with GDF-15 in neural progenitor cells, retinal pigment epithelial cells, and lens epithelial cells and then treated with factors that promote EMT, GDF-11, and TGFβ2, respectively. To further investigate the efficacy of GDF-15 on alleviating lens opacity, we used mouse lens explant culture to mimic secondary cataracts. We pretreated the lens culture with GDF-15 and then added TGFβ2 to develop lens opacity (n = 3 for each group). Western blot and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to measure EMT protein and gene expression, respectively.
RESULTS
In cell culture, GDF-15 pretreatment significantly attenuated EMT marker expression in cultured cells induced by treatment with GDF-11 or TGFβ2. In the lens explant culture, GDF-15 pretreatment also reduced mouse lens opacity induced by exposure to TGFβ2.
CONCLUSIONS
Our results indicate that GDF-15 could alleviate TGFβ2-induced EMT and is a potential therapeutic agent to slow or prevent posterior capsular opacification (PCO) progression after cataract surgery.
TRANSLATIONAL RELEVANCE
Cataracts are the leading cause of blindness worldwide, with the only current treatment involving surgical removal of the lens and replacement with an artificial lens. However, PCO, also known as secondary cataract, is a common complication after cataract surgery. The development of an adjuvant that slows the progression of PCO will be beneficial to the field of anterior complications.
Topics: Animals; Epithelial-Mesenchymal Transition; Transforming Growth Factor beta2; Growth Differentiation Factor 15; Cataract; Mice; Lens, Crystalline; Mice, Inbred C57BL; Cells, Cultured; Disease Models, Animal; Epithelial Cells; Blotting, Western; Retinal Pigment Epithelium
PubMed: 38949633
DOI: 10.1167/tvst.13.7.2 -
Investigative Ophthalmology & Visual... Jul 2024Glucocorticoid-induced glaucoma (GIG) is a prevalent complication associated with glucocorticoids (GCs), resulting in irreversible blindness. GIG is characterized by the...
PURPOSE
Glucocorticoid-induced glaucoma (GIG) is a prevalent complication associated with glucocorticoids (GCs), resulting in irreversible blindness. GIG is characterized by the abnormal deposition of extracellular matrix (ECM) in the trabecular meshwork (TM), elevation of intraocular pressure (IOP), and loss of retinal ganglion cells (RGCs). The objective of this study is to investigate the effects of nicotinamide riboside (NR) on TM in GIG.
METHODS
Primary human TM cells (pHTMs) and C57BL/6J mice responsive to GCs were utilized to establish in vitro and in vivo GIG models, respectively. The study assessed the expression of ECM-related proteins in TM and the functions of pHTMs to reflect the effects of NR. Mitochondrial morphology and function were also examined in the GIG cell model. GIG progression was monitored through IOP, RGCs, and mitochondrial morphology. Intracellular nicotinamide adenine dinucleotide (NAD+) levels of pHTMs were enzymatically assayed.
RESULTS
NR significantly prevented the expression of ECM-related proteins and alleviated dysfunction in pHTMs after dexamethasone treatment. Importantly, NR protected damaged ATP synthesis, preventing overexpression of mitochondrial reactive oxygen species (ROS), and also protect against decreased mitochondrial membrane potential induced by GCs in vitro. In the GIG mouse model, NR partially prevented the elevation of IOP and the loss of RGCs. Furthermore, NR effectively suppressed the excessive expression of ECM-associated proteins and mitigated mitochondrial damage in vivo.
CONCLUSIONS
Based on the results, NR effectively enhances intracellular levels of NAD+, thereby mitigating abnormal ECM deposition and TM dysfunction in GIG by attenuating mitochondrial damage induced by GCs. Thus, NR has promising potential as a therapeutic candidate for GIG treatment.
Topics: Animals; Niacinamide; Pyridinium Compounds; Glucocorticoids; Mice, Inbred C57BL; Mitochondria; Mice; Glaucoma; Extracellular Matrix; Intraocular Pressure; Humans; Disease Models, Animal; Trabecular Meshwork; Cells, Cultured; Retinal Ganglion Cells; Reactive Oxygen Species; Dexamethasone; Male
PubMed: 38949632
DOI: 10.1167/iovs.65.8.1 -
BJS Open Jan 2024Textbook outcomes are composite outcome measures that reflect the ideal overall experience for patients. There are many of these in the elective surgery literature but...
BACKGROUND
Textbook outcomes are composite outcome measures that reflect the ideal overall experience for patients. There are many of these in the elective surgery literature but no textbook outcomes have been proposed for patients following emergency laparotomy. The aim was to achieve international consensus amongst experts and patients for the best Textbook Outcomes for non-trauma and trauma emergency laparotomy.
METHODS
A modified Delphi exercise was undertaken with three planned rounds to achieve consensus regarding the best Textbook Outcomes based on the category, number and importance (Likert scale of 1-5) of individual outcome measures. There were separate questions for non-trauma and trauma. A patient engagement exercise was undertaken after round 2 to inform the final round.
RESULTS
A total of 337 participants from 53 countries participated in all three rounds of the exercise. The final Textbook Outcomes were divided into 'early' and 'longer-term'. For non-trauma patients the proposed early Textbook Outcome was 'Discharged from hospital without serious postoperative complications (Clavien-Dindo ≥ grade III; including intra-abdominal sepsis, organ failure, unplanned re-operation or death). For trauma patients it was 'Discharged from hospital without unexpected transfusion after haemostasis, and no serious postoperative complications (adapted Clavien-Dindo for trauma ≥ grade III; including intra-abdominal sepsis, organ failure, unplanned re-operation on or death)'. The longer-term Textbook Outcome for both non-trauma and trauma was 'Achieved the early Textbook Outcome, and restoration of baseline quality of life at 1 year'.
CONCLUSION
Early and longer-term Textbook Outcomes have been agreed by an international consensus of experts for non-trauma and trauma emergency laparotomy. These now require clinical validation with patient data.
Topics: Humans; Laparotomy; Delphi Technique; Postoperative Complications; Consensus; Emergencies; Outcome Assessment, Health Care
PubMed: 38949628
DOI: 10.1093/bjsopen/zrad145