ER proteins decipher the tubulin code to regulate organelle distribution

Organelles move along differentially modified microtubules to establish and maintain their proper distributions and functions1,2. However, how cells interpret these post-translational microtubule modification codes to selectively regulate organelle positioning remains largely unknown. The endoplasmic reticulum (ER) is an interconnected network of diverse morphologies that extends promiscuously throughout the cytoplasm3, forming abundant contacts with other organelles4. Dysregulation of endoplasmic reticulum morphology is tightly linked to neurologic disorders and cancer5,6. Here we demonstrate that three membrane-bound endoplasmic reticulum proteins preferentially interact with different microtubule populations, with CLIMP63 binding centrosome microtubules, kinectin (KTN1) binding perinuclear polyglutamylated microtubules, and p180 binding glutamylated microtubules. Knockout of these proteins or manipulation of microtubule populations and glutamylation status results in marked changes in endoplasmic reticulum positioning, leading to similar redistributions of other organelles. During nutrient starvation, cells modulate CLIMP63 protein levels and p180–microtubule binding to bidirectionally move endoplasmic reticulum and lysosomes for proper autophagic responses.

Coordinating Carbon Metabolism and Cell Cycle of Chlamydomonasreinhardtii with Light Strategies under Nitrogen Recovery

Nutrient supplementation is common in microalgae cultivation to enhance the accumulation of biomass and biofunctional products, while the recovery mechanism from nutrient starvation is less investigated. In this study, the influence of remodeled carbon metabolism on cell cycle progression was explored by using different light wavelengths under N-repletion and N-recovery. The results suggested that blue light enhanced cell enlargement and red light promoted cell division under N-repletion. On the contrary, blue light promoted cell division by stimulating cell cycle progression under N-recovery. This interesting phenomenon was ascribed to different carbon metabolisms under N-repletion and N-recovery. Blue light promoted the recovery of photosystem II and redirected carbon skeletons into proteins under N-recovery, which potentially accelerated cell recovery and cell cycle progression. Although red light also facilitated the recovery of photosystem II, it mitigated the degradation of polysaccharide and then arrested almost all the cells in the G1 phase. By converting light wavelengths at the 12 h of N-recovery with blue light, red and white lights were proved to increase biomass concentration better than continuous blue light. These results revealed different mechanisms of cell metabolism of Chlamydomonas reinhardtii during N-recovery and could be applied to enhance cell vitality of microalgae from nutrient starvation and boost biomass production.

Seleno-amino acids: A novel class of anti-tuberculosis compounds identified through modified culture screening conditions

<p>Tuberculosis continues to be a major world health problem, causing more deaths than any other bacterial disease. Long treatment durations using a complex cocktail of drugs are often associated with patient non-adherence to therapy, and this has accelerated the development of drug resistant strains. Tuberculosis drug resistance has developed to the extent that some strains are resistant to all clinically used drugs. Therefore novel tuberculosis treatment drugs are urgently required to combat these resistant strains, sterilise latent infections and reduce lengthy treatment durations. This research developed and optimised a high-throughput assay to screen chemical libraries for compounds with anti-mycobacterial activity. The assay utilised fast growing tuberculosis model species M. smegmatis expressing foreign green fluorescent protein (GFP). GFP allowed bacterial growth inhibition to be measured both by fluorescence in addition to absorbance. The assay was expanded to four different culture conditions two of which were nutrient starvation that better mimicked environmental conditions M. tuberculosis is exposed to during infection. These differential culture conditions also revealed previously unidentified mycobacterial inhibitors. Three chemical libraries totaling over 5,000 compounds were screened in the different culture conditions. Seleno-amino acids (Se-AAs), a novel class of anti-tuberculosis compounds, were discovered through screens in nutrient starvation conditions. Based on traits of strong inhibitory activity towards mycobacteria, low human cell line cytotoxicity, structural novelty and known over-the-counter sale as a non-prescription dietary supplement, the Se-AAs were chosen as a promising pharmacophore for further study. Using evidence derived from anti-sense gene knockdown, transposon mutagenesis and biochemical enzyme assays, a pro-drug hypothesis of anti-mycobacterial activity was proposed that involved Se-AAs being transported into the mycobacterial cell by nutrient uptake transporters and subsequent cleavage into catalytically active methylselenium species by lyase enzymes used in mycobacterial sulphurous amino acid metabolism. The activated methylselenium is reduced by mycobacterial redox homeostasis enzymes involved in mycobacterial oxidative defence such as alkyl hydroperoxidases, generating reactive oxygen radical products that damage mycobacterial DNA, lipids and proteins. Reduced methylselenol can be cycled back to the oxidised state by cellular mycothiones, continuously generating damaging reactive oxygen species within the mycobacterial cell. Methylselenium species also disrupt essential mycobacterial processes, such as ketosteroid catabolism and iron-sulphur cluster protein function. In summary, this research has designed and implemented a novel dual label differential culture condition assay useful in the screening and detection of chemicals with anti-tuberculosis properties. A novel structural class of anti-tuberculosis compounds with therapeutic potential, the Se-AAs, was discovered using this assay, the structure-activity relationship of the Se-AAs was explored and a three-component model of Se-AA anti-tuberculosis activity is proposed.</p>

Seleno-amino acids: A novel class of anti-tuberculosis compounds identified through modified culture screening conditions

<p>Tuberculosis continues to be a major world health problem, causing more deaths than any other bacterial disease. Long treatment durations using a complex cocktail of drugs are often associated with patient non-adherence to therapy, and this has accelerated the development of drug resistant strains. Tuberculosis drug resistance has developed to the extent that some strains are resistant to all clinically used drugs. Therefore novel tuberculosis treatment drugs are urgently required to combat these resistant strains, sterilise latent infections and reduce lengthy treatment durations. This research developed and optimised a high-throughput assay to screen chemical libraries for compounds with anti-mycobacterial activity. The assay utilised fast growing tuberculosis model species M. smegmatis expressing foreign green fluorescent protein (GFP). GFP allowed bacterial growth inhibition to be measured both by fluorescence in addition to absorbance. The assay was expanded to four different culture conditions two of which were nutrient starvation that better mimicked environmental conditions M. tuberculosis is exposed to during infection. These differential culture conditions also revealed previously unidentified mycobacterial inhibitors. Three chemical libraries totaling over 5,000 compounds were screened in the different culture conditions. Seleno-amino acids (Se-AAs), a novel class of anti-tuberculosis compounds, were discovered through screens in nutrient starvation conditions. Based on traits of strong inhibitory activity towards mycobacteria, low human cell line cytotoxicity, structural novelty and known over-the-counter sale as a non-prescription dietary supplement, the Se-AAs were chosen as a promising pharmacophore for further study. Using evidence derived from anti-sense gene knockdown, transposon mutagenesis and biochemical enzyme assays, a pro-drug hypothesis of anti-mycobacterial activity was proposed that involved Se-AAs being transported into the mycobacterial cell by nutrient uptake transporters and subsequent cleavage into catalytically active methylselenium species by lyase enzymes used in mycobacterial sulphurous amino acid metabolism. The activated methylselenium is reduced by mycobacterial redox homeostasis enzymes involved in mycobacterial oxidative defence such as alkyl hydroperoxidases, generating reactive oxygen radical products that damage mycobacterial DNA, lipids and proteins. Reduced methylselenol can be cycled back to the oxidised state by cellular mycothiones, continuously generating damaging reactive oxygen species within the mycobacterial cell. Methylselenium species also disrupt essential mycobacterial processes, such as ketosteroid catabolism and iron-sulphur cluster protein function. In summary, this research has designed and implemented a novel dual label differential culture condition assay useful in the screening and detection of chemicals with anti-tuberculosis properties. A novel structural class of anti-tuberculosis compounds with therapeutic potential, the Se-AAs, was discovered using this assay, the structure-activity relationship of the Se-AAs was explored and a three-component model of Se-AA anti-tuberculosis activity is proposed.</p>

Imaging NAD(H) Redox Alterations in Cryopreserved Alveolar Macrophages from Ozone-Exposed Mice and the Impact of Nutrient Starvation during Long Lag Times

Employing the optical redox imaging technique, we previously identified a significant redox shift of nicotinamide adenine dinucleotide (NAD and the reduced form NADH) in freshly isolated alveolar macrophages (AM) from ozone-exposed mice. The goal here was twofold: (a) to determine the NAD(H) redox shift in cryopreserved AM isolated from ozone-exposed mice and (b) to investigate whether there is a difference in the redox status between cryopreserved and freshly isolated AM. We found: (i) AM from ozone-exposed mice were in a more oxidized redox state compared to that from filtered air (FA)-exposed mice, consistent with the results obtained from freshly isolated mouse AM; (ii) under FA exposure, there was no significant NAD(H) redox difference between fresh AM that had been placed on ice for 2.5 h and cryopreserved AM; however, under ozone exposure, fresh AM were more oxidized than cryopreserved AM; (iii) via the use of nutrient starvation and replenishment and H2O2-induced oxidative stress of an AM cell line, we showed that this redox difference between cryopreserved and freshly isolated AM is likely the result of the double “hit”, i.e., the ozone-induced oxidative stress plus nutrient starvation that prevented freshly isolated AM from a full recovery after being on ice for a prolonged time period. The cryopreservation technique we developed eliminates/minimizes the effects of oxidative stress and nutrient starvation on cells. This method can be adopted to preserve lung macrophages from animal models or clinical patients for further investigations.