Staphylococcus aureus: a blemish on skin immunity

Staphylococcus aureus is the leading cause of human skin infections. In this issue of Cell Host & Microbe, new research probes how a change in surface hydrophobicity mediated by a single S. aureus protein renders the pathogen resistant to key molecular effectors of skin innate immunity, including cationic antimicrobial peptides and fatty acid constituents of sebum. Novel treatment strategies for S. aureus infection may lie in supplementing the very same innate defense molecules to therapeutic levels.     

Manufacturing flexibility and performance: bridging the gap between theory and practice

How firms scan and interpret their environments has implications for the flexibility strategy that they choose, as well as for the performance of that strategy. We extend Daft and Weick’s (Acad Manage Rev 9(2):284–295, 1984) model of firms as interpretation systems into a theoretical model of flexibility performance through extended iterations between observations of a failed flexibility initiative and relevant literature. We test the model using well-known teaching cases. We argue that the use of an iterative process that involves cases and theory both stimulates creativity in integrating theory and lays an initial foundation for evidence-based practice.     

A tandem affinity purification-based technology platform to study the cell cycle interactome in Arabidopsis thaliana

Defining protein complexes is critical to virtually all aspects of cell biology because many cellular processes are regulated by stable protein complexes, and their identification often provides insights into their function. We describe the development and application of a high throughput tandem affinity purification/mass spectrometry platform for cell suspension cultures to analyze cell cycle-related protein complexes in Arabidopsis thaliana. Elucidation of this protein-protein interaction network is essential to fully understand the functional differences between the highly redundant cyclin-dependent kinase/cyclin modules, which are generally accepted to play a central role in cell cycle control, in all eukaryotes. Cell suspension cultures were chosen because they provide an unlimited supply of protein extracts of actively dividing and undifferentiated cells, which is crucial for a systematic study of the cell cycle interactome in the absence of plant development. Here we report the mapping of a protein interaction network around six known core cell cycle proteins by an integrated approach comprising generic Gateway-based vectors with high cloning flexibility, the fast generation of transgenic suspension cultures, tandem affinity purification adapted for plant cells, matrix-assisted laser desorption ionization tandem mass spectrometry, data analysis, and functional assays. We identified 28 new molecular associations and confirmed 14 previously described interactions. This systemic approach provides new insights into the basic cell cycle control mechanisms and is generally applicable to other pathways in plants.     

Screening the pandemic response box identified benzimidazole carbamates,Olorofim and ravuconazole as promising drug candidates for the treatment of eumycetoma

Eumycetoma is a chronic subcutaneous neglected tropical disease that can be caused by more than 40 different fungal causative agents. The most common causative agents produce black grains and belong to the fungal orders Sordariales and Pleosporales. The current antifungal agents used to treat eumycetoma are itraconazole or terbinafine, however, their cure rates are low. To find novel drugs for eumycetoma, we screened 400 diverse drug-like molecules from the Pandemic Response Box against common eumycetoma causative agents as part of the Open Source Mycetoma initiative (MycetOS). 26 compounds were able to inhibit the growth of Madurella mycetomatis, Madurella pseudomycetomatis and Madurella tropicana, 26 compounds inhibited Falciformispora senegalensis and seven inhibited growth of Medicopsis romeroi in vitro. Four compounds were able to inhibit the growth of all five species of fungi tested. They are the benzimidazole carbamates fenbendazole and carbendazim, the 8-aminoquinolone derivative tafenoquine and MMV1578570. Minimal inhibitory concentrations were then determined for the compounds active against M. mycetomatis. Compounds showing potent activity in vitro were further tested in vivo. Fenbendazole, MMV1782387, ravuconazole and olorofim were able to significantly prolong Galleria mellonella larvae survival and are promising candidates to explore in mycetoma treatment and to also serve as scaffolds for medicinal chemistry optimisation in the search for novel antifungals to treat eumycetoma.     

Diverse microbial interactions with the basement membrane barrier

During primary contact with susceptible hosts, microorganisms face an array of barriers that thwart their invasion process. Passage through the basement membrane (BM), a 50-100-nm-thick crucial barrier underlying epithelia and endothelia, is a prerequisite for successful host invasion. Such passage allows pathogens to reach nerve endings or blood vessels in the stroma and to facilitate spread to internal organs. During evolution, several pathogens have developed different mechanisms to cross this dense matrix of sheet-like proteins. To breach the BM, some microorganisms have developed independent mechanisms, others hijack host cells that are able to transverse the BM (e.g. leukocytes and dendritic cells) and oncogenic microorganisms might even trigger metastatic processes in epithelial cells to penetrate the underlying BM.     

Detecting drug-resistant tuberculosis: the importance of rapid testing

Despite numerous intervention strategies, including the direct observed short-course treatment strategy and improved diagnostic methods, the incidence of multidrug-resistant and extensively drug-resistant tuberculosis (TB) continues to rise globally. Many treatment policies are based on the model that acquisition of drug resistance in already infected individuals drives the drug-resistant TB epidemic, hence the focus on drug-resistance testing of retreatment cases. However, molecular epidemiology and mathematical modeling suggest that the majority of multidrug-resistant TB cases are due to ongoing transmission of multidrug-resistant strains. This is most likely the result of diagnostic delay, thereby emphasizing the need for rapid diagnostics and comprehensive contact tracing, as well as active case finding. Current diagnosis of TB in low-income, high-burden regions relies on smear microscopy and clinical signs and symptoms. However, this smear-centered approach has many pitfalls, including low sensitivity in HIV patients and children, the inability of smear to reveal drug-resistance patterns, and the need for sampling on consecutive days. In order to address these limitations, efforts have been made to expand access to Mycobacterium tuberculosis culture and drug susceptibility testing. However, the slow growth rate of the causative agent, M. tuberculosis, contributes to significant diagnostic delay. Molecular-based diagnostic methods, targeting mutations that are known to confirm drug resistance, are capable of significantly reducing diagnostic delay. Two such methods, the line-probe assay and the real-time PCR-based Xpert? MTB/RIF assay, have been described. The latter test shows particular promise for smear-negative and extrapulmonary specimens. This may prove especially useful in settings where co-infection rates with HIV are high. However, since most research focuses on the performance of both of these assays, further investigations need to be done regarding the impact of the routine implementation of these assays on TB control programs and the cost effectiveness thereof.     

Relief of hypoxia by angiogenesis promotes neural stem cell differentiation by targeting glycolysis

Blood vessels are part of the stem cell niche in the developing cerebral cortex, but their in vivo role in controlling the expansion and differentiation of neural stem cells (NSCs) in development has not been studied. Here, we report that relief of hypoxia in the developing cerebral cortex by ingrowth of blood vessels temporo‐spatially coincided with NSC differentiation. Selective perturbation of brain angiogenesis in vessel‐specific Gpr124 null embryos, which prevented the relief from hypoxia, increased NSC expansion at the expense of differentiation. Conversely, exposure to increased oxygen levels rescued NSC differentiation in Gpr124 null embryos and increased it further in WT embryos, suggesting that niche blood vessels regulate NSC differentiation at least in part by providing oxygen. Consistent herewith, hypoxia‐inducible factor (HIF)‐1α levels controlled the switch of NSC expansion to differentiation. Finally, we provide evidence that high glycolytic activity of NSCs is required to prevent their precocious differentiation in vivo. Thus, blood vessel function is required for efficient NSC differentiation in the developing cerebral cortex by providing oxygen and possibly regulating NSC metabolism.     

Carbohydrate metabolism is perturbed in peroxisome-deficient hepatocytes due to mitochondrial dysfunction, AMP-activated protein kinase (AMPK) activation, and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) suppression

Hepatic peroxisomes are essential for lipid conversions that include the formation of mature conjugated bile acids, the degradation of branched chain fatty acids, and the synthesis of docosahexaenoic acid. Through unresolved mechanisms, deletion of functional peroxisomes from mouse hepatocytes (L-Pex5(-/-) mice) causes severe structural and functional abnormalities at the inner mitochondrial membrane. We now demonstrate that the peroxisomal and mitochondrial anomalies trigger energy deficits, as shown by increased AMP/ATP and decreased NAD(+)/NADH ratios. This causes suppression of gluconeogenesis and glycogen synthesis and up-regulation of glycolysis. As a consequence, L-Pex5(-/-) mice combust more carbohydrates resulting in lower body weights despite increased food intake. The perturbation of carbohydrate metabolism does not require a long term adaptation to the absence of functional peroxisomes as similar metabolic changes were also rapidly induced by acute elimination of Pex5 via adenoviral administration of Cre. Despite its marked activation, peroxisome proliferator-activated receptor α (PPARα) was not causally involved in these metabolic perturbations, because all abnormalities still manifested when peroxisomes were eliminated in a peroxisome proliferator-activated receptor α null background. Instead, AMP-activated kinase activation was responsible for the down-regulation of glycogen synthesis and induction of glycolysis. Remarkably, PGC-1α was suppressed despite AMP-activated kinase activation, a paradigm not previously reported, and they jointly contributed to impaired gluconeogenesis. In conclusion, lack of functional peroxisomes from hepatocytes results in marked disturbances of carbohydrate homeostasis, which are consistent with adaptations to an energy deficit. Because this is primarily due to impaired mitochondrial ATP production, these L-Pex5-deficient livers can also be considered as a model for secondary mitochondrial hepatopathies.     

Importance of position 8 in μ-conotoxin KIIIA for voltage-gated sodium channel selectivity

μ-Conotoxin KIIIA from Conus kinoshitai is a 16-residue peptide that acts as a potent pore blocker of several voltage-gated sodium channels (Na(v)). In order to obtain more selective blockers and to investigate the role of Trp at position 8, we substituted this residue with Arg, Gln and Glu. KIIIA and analogues were tested on a range of Na(v) expressed in Xenopus laevis oocytes. The rank order of potency for KIIIA was: rNa(v)1.4 ≥ rNa(v)1.2 > mNa(v)1.6 > rNa(v)1.3, with IC(50) values of 48 ± 6 nm, 61 ± 5 nm, 183 ± 31 nm and 3.6 ± 0.3 μm, respectively, whereas no effect was seen on hNa(v)1.5 and hNa(v)1.8 at a concentration of 10 μm. Replacement of Trp8 resulted in more selective blockers with a preference for neuronal sodium channels over the skeletal sodium channel. The activity on rNa(v)1.4 was reduced about 40-, 70- and 200-fold for [W8R]KIIIA, [W8Q]KIIIA and [W8E]KIIIA, respectively. All analogues showed a completely reversible block of rNa(v)1.2, as opposed to the partial reversibility of KIIIA. At saturating concentrations, complete block of rNa(v)1.2 was never achieved. The residual current was lower than 10%, except for [W8E]KIIIA. KIIIA had no effect on the voltage dependence of activation of rNa(v)1.2, whereas all analogues caused a depolarizing shift. Overall, this study shows that Trp8 is a key residue in the pharmacophore. Replacement of Trp8 enables more selective blockers to be obtained for neuronal sodium channels. Trp is a key determinant for the reversibility of block of rNa(v)1.2.