Antimicrobial spiroketal macrolides and dichloro-diketopiperazine from Micromonospora sp. FIMYZ51.

Four compounds (1-4) featuring with an L-rhodinose and spiroketal, possess uncommon continuous hydroxy groups in the macrolide skeleton, and a dichloro-diketopiperazine (5) were isolated from a marine derived Micromonospora sp. FIMYZ51. The determination of the relative and absolute configurations of all isolates was achieved by extensive spectroscopic analyses, single-crystal X-ray diffraction analysis, and ECD calculations. According to structural characteristic and genomic sequences, a plausible biosynthetic pathway for compound 1-4 was proposed and a spirocyclase was inferred to be responsible for the formation of the rare spirocyclic moiety. Compounds 1-4 exhibited potent antifungal activities which is equal to itraconazole against Aspergillus niger. Compounds 1-5 exhibited different degree of inhibitory activities against opportunistic pathogenic bacteria of endocarditis (Micrococcus luteus) with MIC values ranging from 0.0625 µg/mL to 32 µg/mL. Compounds 2 and 3 showed moderate cytotoxicity against drug-resistant tumor cell lines (Namalwa and U266). The result not only provides active lead-compounds, but also reveal the potential of the spirocyclase gene resources from Micromonospora sp., which highlights the promising potential of the strain for biomedical applications.

Regional differences in susceptibility to spinosyn insecticides registered for Colorado potato beetle management in Canada.

The Colorado potato beetle (CPB) is the most economically important pest of Canadian potato, and if left uncontrolled, it can completely consume the crop. In the past decade, the control of CPB has relied heavily on systemic insecticides, principally the neonicotinoids thiamethoxam and clothianidin. Resistance to neonicotinoids in CPB has been well documented in the past 2 decades and mechanisms underlying the resistance better understood. In contrast, resistance to other insecticide classes, including spinosyns (spinosad and spinetoram) and anthranillic diamides (chlorantraniliprole and cyantraniliprole), have not been studied to the same degree in CPB. Spinosyns are the only insecticide certified for organic potato growers in Canada and are frequently applied as a mid-season foliar spray by conventional growers when seed treatments with neoniconitoid or diamide experience control breaks. Improved knowledge on resistance to spinosyns in CPB would allow for the development of regional management strategies. A survey of insecticide susceptibility in CPB populations from 6 potato growing regions between 2018 and 2022 observed: 1) spatial and temporal resistance trends; 2) cross-resistance; and 3) evidence of regional differences in susceptibility to spinosyns. The proportion of populations within each province considered resistant to spinosyns was, in descending order: Québec (16%) > Ontario (14%) > Manitoba (13%) > New Brunswick (9%) > Prince Edward Island (2%) > Alberta (0%). There was a significant change in CPB mortality at the diagnostic concentration (DC = LC90) for spinosad and spinetoram in the 6 provinces but only for year 5 relative to the previous 4 years. Moderate cross-resistance was determined between spinosad and spinetoram with the DC mortality for all populations based on a positive and significant correlation (adjusted R2 = 0.3758; P = 1.263e-13). There was also a positive relationship observed between the number of spinosyn applications (years applied at the sampling location) and declining susceptibility to spinosad (R2 = 0.0927; P < 0.002). Cross-resistance was observed between spinosyns and insecticides in the other two classes, the more significant correlation was between spinosad and tetraniliprole (R2 = 0.3025; P < 0.0002). In Québec, the greater spinosad use in organic potato farms led to resistance in those CPB populations, but spinosyn resistance at conventional farms was not related to greater application of neonicotinoids and diamides. Spinosyns remain relatively effective, nevertheless growers should be concerned over the increasing cases of reduced susceptibility in conventional potato farms and resistance where organic production occurs. Resistance management should continue to encourage rotation with products from the other classes in season and between years in order to extend spinosyn use for CPB control.

gyrA Mutations in Mycoplasma genitalium and Their Contribution to Moxifloxacin Failure: Time for the Next Generation of Resistance-Guided Therapy.

BACKGROUND: Although single nucleotide polymorphisms (SNPs) in Mycoplasma genitalium parC contribute to fluoroquinolone treatment failure, data are limited for the homologous gene, gyrA. This study investigated the prevalence of gyrA SNPs and their contribution to fluoroquinolone failure. METHODS: Samples from 411 patients (male and female) undergoing treatment for M. genitalium infection (Melbourne Sexual Health Centre, March 2019-February 2020) were analyzed by Sanger sequencing (gyrA and parC). For patients treated with moxifloxacin (n = 194), the association between SNPs and microbiologic treatment outcome was analyzed. RESULTS: The most common parC SNP was G248T/S83I (21.1% of samples), followed by D87N (2.3%). The most common gyrA SNP was G285A/M95I (7.1%). Dual parC/gyrA SNPs were found in 8.6% of cases. One third of infections harboring parC G248T/S83I SNP had a concurrent SNP in gyrA conferring M95I. SNPs in gyrA cooccurred with parC S83I variations. Treatment failure was higher in patients with parC S83I/gyrA dual SNPs when compared with infections with single S83I SNP alone from analysis of (1) 194 cases in this study (81.2% vs 45.8%, P = .047), and (2) pooled analysis of a larger population of 535 cases (80.6% vs 43.2%; P = .0027), indicating a strong additive effect. CONCLUSIONS: Compared with parC S83I SNP alone, M. genitalium infections with dual mutations affecting parC/gyrA had twice the likelihood of failing moxifloxacin. Although antimicrobial resistance varies by region globally, these data indicate that gyrA should be considered as a target for future resistance assays in Australasia. We propose a strategy for the next generation of resistance-guided therapy incorporating parC and gyrA testing.

Macrolide therapy in Pseudomonas aeruginosa infections causes uL4 ribosomal protein mutations leading to high-level resistance.

OBJECTIVES: Pseudomonas aeruginosa colonizes the cystic fibrosis (CF) airways causing chronic bacterial lung infections. CF patients are routinely treated with macrolides, however, P. aeruginosa is considered insusceptible as consequence of inadequate susceptibility testing leaving resistance mechanism completely overlooked. Here, we investigated a new mechanism of macrolide resistance caused by ribosomal protein mutations. METHODS: Investigating a longitudinal collection of 529 isolates from CF patients and analysing 5758 protein sequences from different sources, mutations in P. aeruginosa's ribosomal proteins connected to macrolide resistance were identified. Using a modified susceptibility testing protocol, isolates harbouring a mutated uL4 ribosomal protein were tested for resistance against macrolide antibiotics and macrolide-induced quorum sensing modulation. Proteome and ribosome profiling were applied to assess the impact of the mutations on the bacterial physiology. RESULTS: Five uL4 mutations were identified in isolates from different CF patients. Most mapped to the conserved loop region of uL4 and resulted in increased macrolide tolerance (>10-fold relative to wt strains). Greater concentrations (>10-fold) of macrolide antibiotic were needed to inhibit the growth, reduce swimming motility, and induce redox sensitivity of the uL4 mutants. 16 proteins involved in ribosome adaptation displayed altered expression possibly to compensate for the uL4 mutations, which changed the ribosome stoichiometry without negatively affecting bacterial physiology. CONCLUSIONS: Macrolide antibiotics should, therefore, be considered as active antimicrobial agents against P. aeruginosa and resistance development should be contemplated when patients are treated with prolonged courses of macrolides. Importantly, improved macrolide susceptibility testing is necessary for the detection of resistant bacteria.

Increased Prevalence of Minocycline-Resistant Staphylococcus epidermidis with tet(M) by Tetracycline Use for Acne Treatment.

Staphylococcus epidermidis, a major skin bacterium, can cause opportunistic infections. Use of antimicrobial agents against Cutibacterium acnes for acne treatment becomes a risk factor for emergence of antimicrobial-resistant skin bacteria. In this study, the impact of antimicrobial treatment of acne vulgaris on S. epidermidis antimicrobial resistance was assessed. A total of 344 S. epidermidis strains isolated from patients with acne vulgaris who visited hospital (165 strains) and dermatological clinics (179 strains), respectively, were analyzed. Except for doxycycline, the resistance rates were higher in strains isolated from patients who had used antimicrobials for acne treatment than in those isolated from patients who had not used antimicrobials. The prevalence rates of strains with erm(C) from patients who used macrolides and clindamycin (hospital, 78.0%; clinics, 61.3%) and those of strains with tet(M) from patients who used tetracyclines (hospital, 27.5%; clinics, 42.4%) were significantly higher than those of strains from patients who did not use antimicrobials (p < 0.05). All strains with erm(A) (8/8) and 91.7% strains with erm(C) (156/170) showed high-level resistance to macrolides and clindamycin (MIC ≥256 µg/mL). Furthermore, almost all strains with tet(M) showed resistance to minocycline. Our results showed that the use of antimicrobials for acne treatment may lead to an increased prevalence of antimicrobial-resistant S. epidermidis. In particular, the emergence of minocycline-resistant strains with tet(M) owing to the use of tetracyclines (doxycycline and minocycline) is a critical issue. Appropriate antimicrobial use for acne treatment may be an important strategy to prevent the emergence of antimicrobial-resistant skin bacteria.

C. acnes qPCR-Based Antibiotics Resistance Assay (ACQUIRE) Reveals Widespread Macrolide Resistance in Acne Patients and Can Eliminate Macrolide Misuse in Acne Treatment.

Background: Macrolides have been widely used to treat moderate-to-severe acne for more than 50 years. However, the prevalent antibiotic resistance of Propionibacterium acnes, along with the absence of clinically available resistance tests, has made macrolide misuse a frequent occurrence around the globe, with serious consequences. Objective: We developed Cutibacterium acnes quantitative PCR (qPCR)-based antibiotics resistance assay (ACQUIRE) to enable fast and accurate detection of C. acnes macrolide resistance in clinical settings, representing an opportunity to administer antibiotics more wisely and improve the quality of care. Methods: A cross-sectional observational study (n = 915) was conducted to probe into the macrolide resistance of C. acnes in patients with acne. Results: The high sensitivity of ACQUIRE enabled us to reveal a much higher C. acnes 23S recombinant DNA (rDNA) point mutation rate (52%) and thus a higher macrolide resistance (75.5%) compared to previous reports. Carriage of ermX gene was discovered on 472 (53%) subjects, which concurs with previous studies. Conclusion: The macrolide resistance of C. acnes is much higher than previously reported. Integrating ACQUIRE into acne treatment modalities may eliminate macrolide misuse and achieve better clinical improvements.

Calcium-Phosphate Combination Enhances Spinosad Production in Saccharopolyspora spinosa via Regulation of Fatty Acid Metabolism.

Phosphate concentration above 10 mM reduces the production of many secondary metabolites; however, the phenomenon is not mechanistically understood yet. Specifically, the problem of phosphorus limitation in antibiotic production remains unresolved. This study investigates the phosphorus inhibition effect on spinosad production and alleviates it by calcium and phosphate supplementation to fermentation media. Furthermore, we examined the mechanism of fatty acids-induced increase in polyketides production. Four phosphates that were supplemented into the fermentation media include NaH2PO4, Na2HPO4, KH2PO4, and K2HPO4 and NaH2PO4 was found to be the most effective phosphate. Under the optimal phosphate condition of supplementing 20 mM NaH2PO4 on the fourth day and 5 g/L CaCO3, the maximal spinosad production reached 520 mg/L, showing a 1.65-fold increase over the control treatment. In the NaH2PO4-CaCO3 system, the de novo fatty acid biosynthesis was significantly downregulated while spinosad biosynthesis and ß-oxidation were upregulated. The coordination of de novo fatty acid biosynthesis and ß-oxidation promoted intracellular acetyl-CoA concentration. The results demonstrate that NaH2PO4-CaCO3 combined addition is a simple and effective strategy to alleviate phosphorus inhibition effect through the regulation of fatty acid metabolism and accumulation of immediate precursors. This information improves our understanding of phosphates' influence on the large-scale production of polyketides.

Use of Molecular Methods To Detect Shigella and Infer Phenotypic Resistance in a Shigella Treatment Study.

Molecular diagnostic methods improve the detection of Shigella, yet their ability to detect Shigella drug resistance on direct stool specimens is less clear. We tested 673 stool specimens from a Shigella treatment study in Bangladesh, including 154 culture-positive stool specimens and their paired Shigella isolates. We utilized a TaqMan array card that included quantitative PCR (qPCR) assays for 24 enteropathogens and 36 antimicrobial resistance (AMR) genes. Shigella was detected by culture in 23% of stool specimens (154/673), while qPCR detected Shigella at diarrhea-associated quantities in 49% (329/673; P < 0.05). qPCR for AMR genes on the Shigella isolates yielded >94% sensitivity and specificity compared with the phenotypic susceptibility results for azithromycin and ampicillin. The performance for trimethoprim-sulfamethoxazole susceptibility was less robust, and the assessment of ciprofloxacin was limited because most isolates were resistant. The detection of AMR genes in direct stool specimens generally yielded low specificities for predicting the resistance of the paired isolate, whereas the sensitivity and negative predictive values for predicting susceptibility were often higher. For example, the detection of ermB or mphA in stool yielded a specificity of 56% but a sensitivity of 91% and a negative predictive value of 91% versus the paired isolate's azithromycin resistance result. Patients who received azithromycin prior to presentation were universally culture negative (0/112); however, qPCR still detected Shigella at diarrhea-associated quantities in 34/112 (30%). In sum, molecular diagnostics on direct stool specimens greatly increase the diagnostic yield for Shigella, including in the setting of prior antibiotics. The molecular detection of drug resistance genes in direct stool specimens had low specificity for confirming resistance but could potentially "rule out" macrolide resistance.

Preclinical evaluation of strasseriolides A-D, potent antiplasmodial macrolides isolated from Strasseria geniculata CF-247,251.

BACKGROUND: Malaria is a global health problem for which novel therapeutic compounds are needed. To this end, a recently published novel family of antiplasmodial macrolides, strasseriolides A-D, was herein subjected to in vivo efficacy studies and preclinical evaluation in order to identify the most promising candidate(s) for further development. METHODS: Preclinical evaluation of strasseriolides A-D was performed by MTT-based cytotoxicity assay in THLE-2 (CRL-2706) liver cells, cardiotoxicity screening using the FluxOR™ potassium assay in hERG expressed HEK cells, LC-MS-based analysis of drug-drug interaction involving CYP3A4, CYP2D6 and CYP2C9 isoforms inhibition and metabolic stability assays in human liver microsomes. Mice in vivo toxicity studies were also accomplished by i.v. administration of the compounds (vehicle: 0.5% HPMC, 0.5% Tween 80, 0.5% Benzyl alcohol) in mice at 25 mg/kg dosage. Plasma were prepared from mice blood samples obtained at different time points (over a 24-h period), and analysed by LC-MS to quantify compounds. The most promising compounds, strasseriolides C and D, were subjected to a preliminary in vivo efficacy study in which transgenic GFP-luciferase expressing Plasmodium berghei strain ANKA-infected Swiss Webster female mice (n = 4-5) were treated 48 h post-infection with an i.p. dosage of strasseriolide C at 50 mg/kg and strasseriolide D at 22 mg/kg for four days after which luciferase activity was quantified on day 5 in an IVIS® Lumina II imager. RESULTS: Strasseriolides A-D showed no cytotoxicity, no carditoxicity and no drug-drug interaction problems in vitro with varying intrinsic clearance (CLint). Only strasseriolide B was highly toxic to mice in vivo (even at 1 mg/kg i.v. dosage) and, therefore, discontinued in further in vivo studies. Strasseriolide D showed statistically significant activity in vivo giving rise to lower parasitaemia levels (70% lower) compared to the controls treated with vehicle. CONCLUSIONS: Animal efficacy and preclinical evaluation of the recently discovered potent antiplasmodial macrolides, strasseriolides A-D, led to the identification of strasseriolide D as the most promising compound for further development. Future studies dealing on structure optimization, formulation and establishment of optimal in vivo dosage explorations of this novel compound class could enhance their clinical potency and allow for progress to later stages of the developmental pipeline.

Development and characterization of a new swine model of invasive pneumococcal pneumonia.

Streptococcus pneumoniae is the most common microbial cause of community-acquired pneumonia. Currently, there are no available models of severe pneumococcal pneumonia in mechanically ventilated animals to mimic clinical conditions of critically ill patients. We studied endogenous pulmonary flora in 4 healthy pigs and in an additional 10 pigs in which we intra-bronchially instilled S. pneumoniae serotype 19 A, characterized by its resistance to penicillin, macrolides and tetracyclines. The pigs underwent ventilation for 72 h. All pigs that were not challenged with S. pneumoniae completed the 72-h study, whereas 30% of infected pigs did not. At 24 h, we clinically confirmed pneumonia in the infected pigs; upon necropsy, we sampled lung tissue for microbiological/histological confirmation of pneumococcal pneumonia. In control pigs, Streptococcus suis and Staphylococcus aureus were the most commonly encountered pathogens, and their lung tissue mean ± s.e.m. concentration was 7.94 ± 20 c.f.u./g. In infected pigs, S. pneumoniae was found in the lungs of all pigs (mean ± s.e.m. pulmonary concentration of 1.26 × 105 ± 2 × 102 c.f.u./g). Bacteremia was found in 50% of infected pigs. Pneumococcal pneumonia was confirmed in all infected pigs at 24 h. Pneumonia was associated with thrombocytopenia, an increase in prothrombin time, cardiac output and vasopressor dependency index and a decrease in systemic vascular resistance. Upon necropsy, microbiological/histological pneumococcal pneumonia was confirmed in 8 of 10 pigs. We have therefore developed a novel model of penicillin- and macrolide-resistant pneumococcal pneumonia in mechanically ventilated pigs with bacteremia and severe hemodynamic compromise. The model could prove valuable for appraising the pathogenesis of pneumococcal pneumonia, the effects associated with macrolide resistance and the outcomes related to the use of new diagnostic strategies and antibiotic or complementary therapies.

Chitosan Augments Tetramycin against Soft Rot in Kiwifruit and Enhances Its Improvement for Kiwifruit Growth, Quality and Aroma.

In this study, the co-application of chitosan and tetramycin against kiwifruit soft rot and its effects on the disease resistance, growth, quality and aroma of kiwifruit were investigated. The results show that chitosan could effectively enhance tetramycin against soft rot of kiwifruit with the field control efficacy of 85.33% for spraying chitosan 100 time + 0.3% tetramycin AS 5000-time dilution liquid, which was higher than 80.99% for 0.3% tetramycin AS 5000-time dilution liquid and significantly (p < 0.01) higher than 40.66% for chitosan 100-time dilution liquid. Chitosan could significantly (p < 0.05) improve the promoting effects of tetramycin on total phenolics, total flavonoids, SOD activity of kiwifruit compared to tetramycin during storage for 0-28 days and enhance the disease resistance of kiwifruit. Moreover, the co-application of chitosan and tetramycin was more effective than tetramycin or chitosan alone in enhancing fruit growth, improving fruit quality and increasing fruit aroma. This study highlights that chitosan can be used as an adjuvant to enhance tetramycin against soft rot of kiwifruit and promote tetramycin's improvement for the single fruit volume and weight, vitamin C, soluble sugar, soluble solid, dry matter, soluble protein, titratable acidity and aroma of kiwifruit.

Callyspongiolide kills cells by inducing mitochondrial dysfunction via cellular iron depletion.

The highly cytotoxic marine natural product callyspongiolide holds great promise as a warhead of antibody-drug conjugate in cancer therapeutics; however, the mechanism underlying its cytotoxicity remains unclear. To elucidate how callyspongiolide kills cells, we employed label-free target identification with thermal stability-shift-based fluorescence difference in two-dimensional (2-D) gel electrophoresis (TS-FITGE), which allowed observation of a unique phenomenon of protein-spot separation on 2-D gels upon treatment with callyspongiolide at increasing temperatures. During our exploration of what proteins were associated with this phenomenon as well as why it happens, we found that callyspongiolide induces mitochondrial/lysosomal dysfunction and autophagy inhibition. Moreover, molecular biology studies revealed that callyspongiolide causes lysosomal dysfunction, which induces cellular iron depletion and leads to mitochondrial dysfunction and subsequent cytotoxicity. Notably, these effects were rescued through iron supplementation. Although our approach was unable to reveal the direct protein targets of callyspongiolide, unique phenomena observed only by TS-FITGE provided critical insight into the mechanism of action of callyspongiolide and specifically its cytotoxic activity via induction of mitochondrial dysfunction through cellular iron depletion caused by lysosomal deacidification, which occurred independent of known programmed cell death pathways.

Complex effects of macrolide venturicidins on bacterial F-ATPases likely contribute to their action as antibiotic adjuvants.

Bacterial energy metabolism is now recognized as a critical factor for the efficacy of antibiotics. The F-type ATPase/ATP synthase (FOF1) is a central player in cellular bioenergetics of bacteria and eukaryotes, and its potential as a selective antibiotic target has been confirmed by the success of bedaquiline in combatting multidrug-resistant tuberculosis. Venturicidin macrolides were initially identified for their antifungal properties and were found to specifically inhibit FOF1 of eukaryotes and bacteria. Venturicidins alone are not effective antibacterials but recently were found to have adjuvant activity, potentiating the efficacy of aminoglycoside antibiotics against several species of resistant bacteria. Here we discovered more complex effects of venturicidins on the ATPase activity of FOF1 in bacterial membranes from Escherichia coli and Pseudomonas aeruginosa. Our major finding is that higher concentrations of venturicidin induce time- and ATP-dependent decoupling of F1-ATPase activity from the venturicidin-inhibited, proton-transporting FO complex. This dysregulated ATPase activity is likely to be a key factor in the depletion of cellular ATP induced by venturicidins in prior studies with P. aeruginosa and Staphylococcus aureus. Further studies of how this functional decoupling occurs could guide development of new antibiotics and/or adjuvants that target the F-type ATPase/ATP synthase.

Elongation factor eEF2 kinase and autophagy jointly promote survival of cancer cells.

Cells within solid tumours can become deprived of nutrients; in order to survive, they need to invoke mechanisms to conserve these resources. Using cancer cells in culture in the absence of key nutrients, we have explored the roles of two potential survival mechanisms, autophagy and elongation factor 2 kinase (eEF2K), which, when activated, inhibits the resource-intensive elongation stage of protein synthesis. Both processes are regulated through the nutrient-sensitive AMP-activated protein kinase and mechanistic target of rapamycin complex 1 signalling pathways. We find that disabling both autophagy and eEF2K strongly compromises the survival of nutrient-deprived lung and breast cancer cells, whereas, for example, knocking out eEF2K alone has little effect. Contrary to some earlier reports, we find no evidence that eEF2K regulates autophagy. Unexpectedly, eEF2K does not facilitate survival of prostate cancer PC3 cells. Thus, eEF2K and autophagy enable survival of certain cell-types in a mutually complementary manner. To explore this further, we generated, by selection, cells which were able to survive nutrient starvation even when autophagy and eEF2K were disabled. Proteome profiling using mass spectrometry revealed that these 'resistant' cells showed lower levels of diverse proteins which are required for energy-consuming processes such as protein and fatty acid synthesis, although different clones of 'resistant cells' appear to adapt in dissimilar ways. Our data provide further information of the ways that human cells cope with nutrient limitation and to understanding of the utility of eEF2K as a potential target in oncology.

Oxyberberine Prevented Lipopolysaccharide-Induced Acute Lung Injury through Inhibition of Mitophagy.

Acute lung injury (ALI) is a serious respiratory syndrome characterized with uncontrolled inflammatory response. Oxyberberine has strong potential for clinical usage since it showed strong anti-inflammatory, antifungal, and antiarrhythmic effects in various diseases. In the present study, we evaluated whether oxyberberine can inhibit lipopolysaccharide- (LPS-) induced ALI in vivo and further evaluated the possible involvement of mitophagy in vitro by using A549 cells, a human lung epithelial cell line. Our in vivo study shows that oxyberberine significantly inhibited LPS-induced lung pathological injury and lung edema, as indicated by the changes in lung wet/dry ratio and total protein levels in the BALF in mice. Moreover, oxyberberine inhibited inflammation, as indicated by the changes of neutrophil accumulation and production of proinflammatory cytokines including tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), and IL-6 in both the lung and bronchoalveolar lavage fluid (BALF) in ALI mice. Our in vitro study shows that LPS significantly decreased the protein level of mitochondrial proteins, including cytochrome c oxidase subunit IV (COX IV), p62, and mitofusin-2 (Mfn2) in A549 cells. In addition, LPS induced significant Parkin1 translocation from cytoplasm to mitochondria. These changes were significantly inhibited by oxyberberine. Notably, the inhibitory effect of oxyberberine was almost totally lost in the presence of lysosome fusion inhibitor bafilomycin A1 (Baf), a mitophagy inhibitor. In conclusion, the present study demonstrated that oxyberberine alleviated LPS-induced inflammation in ALI via inhibition of Parkin-mediated mitophagy.

Comparison of chloroquine-like molecules for lysosomal inhibition and measurement of autophagic flux in the brain.

Measurement of autophagic flux in vivo is critical to understand how autophagy can be used to combat disease. Neurodegenerative diseases have a special relationship with autophagy, which makes measurement of autophagy in the brain a significant research priority. Currently, measurement of autophagic flux is possible through use of transgenic constructs, or application of a lysosomal inhibitor such as chloroquine. Unfortunately, chloroquine is not useful for measuring autophagic flux in the brain and the use of transgenic animals necessitates cross-breeding of transgenic strains and maintenance of lines, which is costly. To find a drug that could block lysosomal function in the brain for the measurement of autophagic flux, we selected compounds from the literature that appeared to have similar properties to chloroquine and tested their ability to inhibit autophagic flux in cell culture and in mice. These chemicals included chloroquine, quinacrine, mefloquine, promazine and trifluoperazine. As expected, chloroquine blocked lysosomal degradation of the autophagic protein LC3B-II in cell culture. Quinacrine also inhibited autophagic flux in cell culture. Other compounds tested were not effective. When injected into mice, chloroquine caused accumulation of LC3B-II in heart tissue, and quinacrine was effective at blocking LC3B-II degradation in male, but not female skeletal muscle. None of the compounds tested were useful for measuring autophagic flux in the brain. During this study we also noted that the vehicle DMSO powerfully up-regulated LC3B-II abundance in tissues. This study shows that chloroquine and quinacrine can both be used to measure autophagic flux in cells, and in some peripheral tissues. However, measurement of flux in the brain using lysosomal inhibitors remains an unresolved research challenge.

Heat shock increases levels of reactive oxygen species, autophagy and apoptosis.

Hyperthermia is a promising anticancer treatment used in combination with radiotherapy and chemotherapy. Temperatures above 41.5 °C are cytotoxic and hyperthermia treatments can target a localized area of the body that has been invaded by a tumor. However, non-lethal temperatures (39-41 °C) can increase cellular defenses, such as heat shock proteins. This adaptive survival response, thermotolerance, can protect cells against subsequent cytotoxic stress such as anticancer treatments and heat shock (>41.5 °C). Autophagy is another survival process that is activated by stress. This study aims to determine whether autophagy can be activated by heat shock at 42 °C, and if this response is mediated by reactive oxygen species (ROS). Autophagy was increased during shorter heating times (<60 min) at 42 °C in cells. Levels of acidic vesicular organelles (AVO) and autophagy proteins Beclin-1, LC3-II/LC-3I, Atg7 and Atg12-Atg5 were increased. Heat shock at 42 °C increased levels of ROS. Increased levels of LC3 and AVOs at 42 °C were inhibited by antioxidants. Therefore, increased autophagy during heat shock at 42 °C (<60 min) was mediated by ROS. Conversely, heat shock at 42 °C for longer times (1-3 h) caused apoptosis and activation of caspases in the mitochondrial, death receptor and endoplasmic reticulum (ER) pathways. Thermotolerant cells, which were developed at 40 °C, were resistant to activation of apoptosis at 42 °C. Autophagy inhibitors 3-methyladenine and bafilomycin sensitized cells to activation of apoptosis by heat shock (42 °C). Improved understanding of autophagy in cellular responses to heat shock could be useful for optimizing the efficacy of hyperthermia in the clinic.

Trichothecene macrolides from the endophytic fungus Paramyrothecium roridum and their cytotoxic activity.

The chemical investigation of the secondary metabolites of Paramyrothecium roridum (homotypic synonym: Myrothecium roridum), an endophytic fungus isolated from the medicinal plant Morinda officinalis, led to the isolation of twelve cytotoxic trichothecene macrolides, including two new ones, named myrothecines H and I. The structures of the new macrolides were elucidated by extensive spectroscopic measurements analyses. In addition, the cytotoxic activities of these compounds were evaluated against SF-268, NCI-H460, and HepG-2 tumor cell lines, and all isolated compounds (1-12) exhibited significant cytotoxic activity with the IC50 ranging from 0.0002-16.2 µM. Moreover, the inhibitory activity of myrothecines H and I was evidenced by inducing phosphorylation of JNK (c-Jun N-terminal protein kinase) protein and the PARP (poly ADP-ribose polymerase) cleavage, and eventually induce apoptosis of HepG-2 cells. The results indicated that myrothecines H and I could be applied as chemotherapeutic agents.

Astrocytes Control Sensory Acuity via Tonic Inhibition in the Thalamus.

Sensory discrimination is essential for survival. However, how sensory information is finely controlled in the brain is not well defined. Here, we show that astrocytes control tactile acuity via tonic inhibition in the thalamus. Mechanistically, diamine oxidase (DAO) and the subsequent aldehyde dehydrogenase 1a1 (Aldh1a1) convert putrescine into GABA, which is released via Best1. The GABA from astrocytes inhibits synaptically evoked firing at the lemniscal synapses to fine-tune the dynamic range of the stimulation-response relationship, the precision of spike timing, and tactile discrimination. Our findings reveal a novel role of astrocytes in the control of sensory acuity through tonic GABA release.

Sesterterpenes and macrolide derivatives from the endophytic fungus Aplosporella javeedii.

Five sesterterpenes (1-5) including two new compounds (1 and 2), as well as a new (6) and a known macrolide (7) were isolated from the endophytic fungus Aplosporella javeedii. The structures of the new compounds were elucidated by analysis of their 1D and 2D NMR and HRMS data as well as by comparison with the literature. Compound 4 and its acetyl derivatives 4a, 4b, 4c which were prepared by acetylation of 4 exhibited moderate cytotoxicity against the mouse lymphoma cell line L5178Y with IC50 values ranging from 6.2 to 12.8 µM, respectively. Moreover, 4a and 4c exhibited also cytotoxicity against human leukemia (Jurkat J16) and lymphoma (Ramos) cell lines. Compound 7 showed strong cytotoxicity against the L5178Y cell line, as well as against human Jurkat J16 and Ramos cells with IC50 values of 0.4, 5.8, and 4.4 µM, respectively. Mechanistic studies indicated that 7 induces apoptotic cell death. In addition, compounds 3, 4 and 7 showed low antibacterial activities against Mycobacterium tuberculosis H37Rv and compound 6 against Staphylococcus aureus, respectively, with MICs of 100 µM. Preliminary structure-activity relationships are discussed.