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Rufomycin Targets ClpC1 Proteolysis in Mycobacterium tuberculosis and M. abscessus
Mary P. Choules, Nina M. Wolf, Hyun Lee, Jeffrey R. Anderson, Edyta M. Grzelak, Yuehong Wang, Rui Ma, Wei Gao, James B. McAlpine, Ying-Yu Jin, Jinhua Cheng, Hanki Lee, Joo-Won Suh, Nguyen Minh Duc, Seungwha Paik, Jin Ho Choe, Eun-Kyeong Jo, Chulhun L. Chang, Jong Seok Lee, Birgit U. Jaki, Guido F. Pauli, Scott G. Franzblau, Sanghyun Cho
Mary P. Choules
Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, USADepartment of Medicinal Chemistry & Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, USA
Nina M. Wolf
Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, USA
Hyun Lee
Department of Medicinal Chemistry & Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, USACenter for Biomolecular Sciences, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, USA
Jeffrey R. Anderson
Edyta M. Grzelak
Yuehong Wang
Rui Ma
Wei Gao
James B. McAlpine
Ying-Yu Jin
Center for Nutraceutical and Pharmaceutical Materials, Myongji University, Cheoin-gu, Gyeonggi-do, Republic of Korea
Jinhua Cheng
Division of Bioscience and Bioinformatics, College of Natural Science, Myongji University, Cheoin-gu, Gyeonggi-do, Republic of Korea
Hanki Lee
Joo-Won Suh
Center for Nutraceutical and Pharmaceutical Materials, Myongji University, Cheoin-gu, Gyeonggi-do, Republic of KoreaDivision of Bioscience and Bioinformatics, College of Natural Science, Myongji University, Cheoin-gu, Gyeonggi-do, Republic of Korea
Nguyen Minh Duc
Seungwha Paik
Department of Microbiology, Chungnam National University School of Medicine, Daejeon, Republic of KoreaInfection Control Convergence Research Center, Chungnam National University School of Medicine, Daejeon, Republic of Korea
Jin Ho Choe
Eun-Kyeong Jo
Chulhun L. Chang
Department of Laboratory Medicine, Pusan National University Yangsan Hospital, Yangsan, Republic of Korea
Jong Seok Lee
International Tuberculosis Research Center, Changwon, Republic of Korea
Birgit U. Jaki
Guido F. Pauli
Scott G. Franzblau
Sanghyun Cho
ClpC1 is an emerging new target for the treatment of Mycobacterium tuberculosis infections, and several cyclic peptides (ecumicin, cyclomarin A, and lassomycin) are known to act on this target. This study identified another group of peptides, the rufomycins (RUFs), as bactericidal to M. tuberculosis through the inhibition of ClpC1 and subsequent modulation of protein degradation of intracellular proteins. Rufomycin I (RUFI) was found to be a potent and selective lead compound for both M. tuberculosis (MIC, 0.02 μM) and Mycobacterium abscessus (MIC, 0.4 μM). Spontaneously generated mutants resistant to RUFI involved seven unique single nucleotide polymorphism (SNP) mutations at three distinct codons within the N-terminal domain of clpC1 (V13, H77, and F80). RUFI also significantly decreased the proteolytic capabilities of the ClpC1/P1/P2 complex to degrade casein, while having no significant effect on the ATPase activity of ClpC1. This represents a marked difference from ecumicin, which inhibits ClpC1 proteolysis but stimulates the ATPase activity, thereby providing evidence that although these peptides share ClpC1 as a macromolecular target, their downstream effects are distinct, likely due to differences in binding.
In 2016, the World Health Organization (WHO) recognized tuberculosis (TB) as the number one single infectious killer worldwide (1), and this status was maintained through 2017 (2), confirming that TB remains a major public health threat. With the continued emergence of multidrug-resistant and extensively drug-resistant strains (MDR and XDR) of Mycobacterium tuberculosis, it is a priority to identify new cellular targets for the treatment of M. tuberculosis infections. An additional growing health threat is infections with nontuberculous mycobacteria (NTM) (3). NTM can cause pulmonary and disseminated infections that affect immunocompromised and immunocompetent patients equally (4). Rapidly growing mycobacteria, including Mycobacterium abscessus, are major pathogens for pulmonary infections caused by NTM infections (5). The most drug-resistant NTM infections are caused by M. abscessus bacteria (6), and there is an urgent need for new drug development to improve the treatment outcomes for NTM diseases (7).
Over the past decade, several compounds have been identified that inhibit ClpC1 (8), including cyclomarin A (CYMA) (9, 10), lassomycin (11), and ecumicin (ECU) (12). The structures of CYMA and ECU are shown in Fig. 1. ClpC1 is currently not targeted for the treatment of TB but has been established as a viable target for drug design (8, 12–14).
Structures of cyclomarin A (CYMA) and ecumicin (ECU).
ClpC1 is the ATP-dependent homologue of the ClpC class of chaperone proteins present in M. tuberculosis (13) and is highly conserved among mycobacteria. Unlike in many other bacteria, ClpC proteins are essential for the viability of mycobacteria, especially M. tuberculosis (15). In M. tuberculosis, ClpC1 associates with the proteolytic domains, ClpP1 and ClpP2, and together they are responsible for waste protein degradation within the cell (16, 17). The proteolytic domains are strictly regulated by ClpC1 and capable of only low levels of unregulated protein degradation without the assistance of ClpC1. Therefore, without functional ClpC1, protein degradation within the cell is reduced or stopped completely. The downstream proteomic and metabolomic effects of ClpC1 perturbation are still being studied; however, two mechanisms have been proposed: (i) the uncoupling of ClpC1 activity from ClpP activity and (ii) the overactivation of ClpC1, leading to uncontrolled protein degradation (18). ECU has been found to trigger the first proposed mechanism by uncoupling ClpC1 from ClpP activity, leading to a decrease in protein degradation.
The goal of this project was to establish the rufomycins (RUFs) (19–22) (Fig. 2), which are also known as the ilamycins (23–25), as another class of compounds that target ClpC1. The RUFs were originally discovered in 1960 and patented by Takeda Chemical Industries. They were later patented with different claims by Eli Lilly and Company in 2000. The biosynthetic pathway was established by Tomita et al. in 2017 (24). The RUFs are a class of cyclic peptides containing seven amino acids and were found to be active against M. tuberculosis and M. abscessus. This report describes the mechanism of action of the RUF group through the inhibition of ClpC1 with the focus on rufomycin I (RUFI). In addition, this study demonstrated that RUFI is active in macrophages, acting against both M. tuberculosis and M. abscessus.
Rufomycin (RUF) analogues. aMIC against M. tuberculosis.
Isolation of rufomycin I.RUFI was isolated from Streptomyces sp. strain MJM3502, determined to be 100% identical to Streptomyces atratus (NRRL B-16927) through classification using the 16S rRNA gene sequence (see the supplemental material). Strain MJM3502 was obtained by the Extract Collection of Useful Microorganisms (ECUM) at Myongji University, Republic of Korea, and was fermented in glucose-soybean starch (GSS) medium (rich medium). The culture medium supernatants were extracted with ethyl acetate and dried. MJM3502 was identified as a hit from the high-throughput screening (HTS) of approximately 7,000 actinomycete cultures as previously discussed (12). Briefly, RUFI was isolated by stepwise bioassay guided fractionation of the MJM3502 extract. Primary fractionation was performed using liquid-liquid separation with a biphasic mixture of dichloromethane (DCM), methanol (MeOH), and H2O. The lower layer was collected and dried prior to further separation using C18 flash chromatography with a gradient of acetonitrile (ACN) and H2O. The active fraction was subjected to preparative chromatography on a C18 silica gel using a 45% isocratic elution with ACN containing 0.1% formic acid (FA).
Rufomycin has potent and selective in vitro activity against M. tuberculosis and M. abscessus.RUFI demonstrates potent and selective activity against M. tuberculosis, with MICs comparable or superior to those of standard first- or second-line drugs for the treatment of TB (Tables 1 and 2). RUFI also maintains activity against monoresistant, MDR, and XDR strains (Fig. 3), indicating that the RUFs most likely have a different molecular target than current drugs used in TB therapy. Additionally, RUFI retains its activity against strains from the five global M. tuberculosis clades representative of clinical TB disease across the world.
MICs of RUFI and anti-TB drugs against M. tuberculosis
Spectrum of activity of RUFI
MIC distribution of RUFI against pan-susceptible (blue), MDR (orange), and XDR (gray) M. tuberculosis strains.
Much like for ECU, the inhibitory activities of RUFI and CYMA appear to be specific to mycobacteria, with no detected activity against Candida albicans, Escherichia coli, or Staphylococcus aureus (Table 2). Unlike ECU and CYMA, RUFI shows activity against all tested mycobacteria. Of special interest is its activity against M. abscessus, one of the more difficult-to-treat mycobacteria (26). RUFI has a clear concentration-dependent bactericidal activity against M. abscessus (MBC of 1.2 μM). The drastic difference in the potencies of the three compounds is most likely attributable to differences in physiochemical properties, but this remains unconfirmed. Moreover, RUFI has concentration- and time-dependent bactericidal activity against M. tuberculosis (minimum bactericidal concentration [MBC] of 0.4 μM) but appears to have a more bacteriostatic effect than that of ECU (Fig. 4). When a more concentrated bacterial inoculum was used, the degree of bacterial killing was reduced; therefore, the activity of RUFI could be bacterial inoculum concentration dependent, as is observed for isoniazid (INH). The difference in observed bacterial concentration from time zero (T0) to day 1 (T1) is most likely due to a lull in bacterial growth upon initial introduction to fresh bacterial media from frozen seed stock. Unfortunately, RUFI showed relatively low activity against nonreplicating cultures of M. tuberculosis (MIC, >10 μM; 75% inhibition at 10 μM).
Rufomycin I (RUFI) has time- and concentration-dependent bactericidal activity against M. tuberculosis. The inoculum concentration was 6.0 × 104 CFU/ml. Error bars represent the SDs of three measurements.
As mycobacterial species are known to be capable of surviving in macrophages, RUFI was tested under these conditions. First, J774.1 cells were infected with M. tuberculosis, followed by RUFI treatment (Fig. 5). In this assay, RUFI was even more potent than rifampin (RIF), which is a clinically used first-line anti-TB drug. RUFI treatment also reduced the CFU of M. abscessus infected in bone marrow-derived macrophages (BMDMs) (Fig. 6). RUFI was as effective as clarithromycin (CLR), which is reported to effectively kill M. abscessus residing in macrophages. In short, significant antimicrobial effects of RUFI were observed in both M. tuberculosis and M. abscessus macrophage infections, indicating that RUFI is a potent antimycobacterial compound that can penetrate macrophages to eliminate intracellular mycobacteria.
Activity of RUFI against M. tuberculosis in murine macrophages. Bars represent CFU prior to treatment (T0), no treatment (T6), and treatment with rifampin (RIF) or RUFI at the indicated concentrations. Values are means ± SDs from six measurements. According to the two-tailed t test, significant differences (P < 0.02 [**]) were observed between the untreated group (T0) and the groups treated with RUFI at 0.48 and 0.098 μM.
Activity of RUFI against M. abscessus in BMDMs. Bars represent CFU on the day of infection (D0), the second day with untreated cells (D2), and the second day for cells treated with clarithromycin (CLR) or RUFI at the indicated concentrations. Values are means ± SDs from six measurements with duplication. According to the two-tailed t test, significant differences (P < 0.001 [**]) were observed between D2 and all treatment groups.
RUFI was also found to act selectively on mycobacteria, as seen with the lack of toxicity to the Vero cell line. Although some toxicity was observed against the macrophage J774.1 cell line (Table 3), the concentration effecting a reduction in fluorescence of 50% relative to untreated cells (IC50) is very similar to that of bedaquiline (BDQ) and far above the MICs for mycobacteria. This gives RUFI a selectivity index (SI) of >2,500 (IC50/MIC) against M. tuberculosis and an SI of >100 against M. abscessus. Interestingly, unlike ECU, RUFI shows some cytotoxic activity against the melanoma cancer cell line MDA-MB-435, the breast cancer cell line MDA-MB-231, and the ovarian cancer cell line OVCAR 3. Studies addressing the mechanism of this cytotoxicity are ongoing and will be reported separately.
Toxicity of RUFI and ECU against mammalian cell lines
Rufomycin-resistant M. tuberculosis strains harbor mutations in clpC1.Spontaneously generated mutants resistant to RUFI (rRUFI) were selected for in vitro testing through stepwise inoculation on 7H11 agar plates impregnated with increasing concentrations of RUFI as described in Materials and Methods. Single-step mutations were selected at a frequency of 2.6 × 10−9. A total of 34 colonies were selected and determined by PCR and Sanger DNA sequencing to contain a single nucleotide polymorphism (SNP) within clpC1 (Table 4). The strains were tested against a panel of standard antibiotics to determine phenotypic specificity to RUFI (Table 1). Based on the level of selective resistance to RUFI, defined as a greater than 4× increase in MIC compared to that for H37Rv, seven strains (strains 28, 31, 35, 41, 50, 51, and 53) containing unique SNP mutations within clpC1 were selected for confirmatory whole-genome sequencing (WGS). All strains were confirmed to harbor a nonsynonymous mutation in clpC1 (Rv3596c) compared to M. tuberculosis H37Rv. Each strain contained one of seven mutations in the N-terminal domain of ClpC1 (10, 12, 13). Similar to what has been reported for ECU (12), multiple mutation sites were identified within the genome of all rRUFI strains relative to H37Rv. Most minor mutations were attributed to genetic drift and therefore determined not to be associated with the molecular target. A few mutations were present in only one of the seven rRUFI strains (53, fadE22 [Rv3061c]; 51, Rv3438, mce4F [Rv349c], and fdxB [Rv3554]; and 35, papA2 [Rv3820c]); however, these mutations were in nonessential genes, and their relation to the function of ClpC1 is unclear at this time. The most common mutations occurred at residue 80, resulting in a replacement of phenylalanine (F) by isoleucine (I), valine (V), serine (S), cysteine (C), or leucine (L). Substitutions of arginine (R) for histidine (H) at residue 77 and phenylalanine for valine at residue 13 were also observed.
Mutations of clpC1 in RUFI-resistant strains
When compared to the spontaneously generated mutations in clpC1 conferring resistance to ECU (L92S, L92F, and L96P) (12) and genetically generated mutants with resistance to CYMA (F2A, F2Y, F80A F80Y, E89A, and E89Q) (10), only alterations of residue 80 were found to be in common. To further test the significance of the observed mutations, ECU, CYMA, and RUFI were tested against M. tuberculosis mutants resisant to RUFI and ECU (Table 1). RUFI and CYMA both maintained their activity against ECU-resistant strains, while ECU also maintained its activity against mutants resistant to RUFI, indicating a lack of cross-resistance. On the other hand, mutants resistant to RUFI harboring the SNP mutation at residue 80 showed resistance to CYMA, while retaining activity against mutants with SNP mutations at residues 77 and 13. The increase from baseline in the MIC of both RUFI and CYMA against M. tuberculosis strains containing a mutation at residue 80, while ECU maintained its full activity, suggests that RUFI might bind to ClpC1 in a fashion similar to that of CYMA but different from that of ECU. This hypothesis is supported by preliminary data indicating antagonistic effects when dosing these compounds in combination, which could be the result of competitive binding effects between the ClpC1 inhibitors.
Rufomycin inhibits proteolytic activity of the ClpP1P2 complex.To clarify the mode of action of RUFI against M. tuberculosis, the compound was subjected to several functional assays previously established to assess the inhibitory capacity of compounds against ClpC1 and the associated proteolytic complex, ClpC1/P1/P2 (Fig. 7) (27–29). The first assay (Fig. 7A) assessed the effect of RUFI on the ATP-dependent ATPase activity of ClpC1. Unlike ECU, which stimulates ATPase activity, resulting in the uncoupling of ATPase activity from proteolytic activity, RUFI had a mild and insignificant inhibitory effect on ClpC1 ATPase activity. This further solidifies the hypothesis that RUFI and ECU affect ClpC1 differently.
ClpC1 ATPase activity (A) and proteolytic activity of the ClpC1/P1/P2 complex (B) in response to ECU and RUFI treatment. This experiment was carried out in triplicate.
The second assay (Fig. 7B) aimed to assess the effect RUFI has on the ability of ClpC1 to stimulate ATP-dependent degradation of the model substrate, fluorescein isothiocyanate (FITC)-casein, by the complex ClpC1/P1/P2. Comparable to ECU, RUFI strongly inhibited the proteolytic capabilities of the ClpC1/P1/P2 complex to degrade casein. This was found to be statistically significant (P < 0.01) at a concentration of 10 μM. The ability of RUFI to inhibit the ATP-dependent selection and subsequent degradation of proteins within the cell can explain the observed cytotoxic effect of RUFI on mycobacteria. This observed inhibitory effect further confirms the cellular target of RUFI and the selectivity of RUFI for mycobacteria.
RUFI binds to mycobacterial ClpC1.RUFI, ECU, and CYMA were tested against M. tuberculosis, M. abscessus, and Mycobacterium chelonae in surface plasmon resonance (SPR) binding assays (Fig. 8; Table 5). ClpC1, especially the N-terminal domain (NTD), is highly conserved among mycobacteria (30). The ClpC1 constructs tested were found to be 100% genetically identical in the NTD but varied by several residues in other regions of the protein. The primary sequences of M. abscessus and M. chelonae have a 99.1% identity, while each has a 91.8% identity to the primary sequence of M. tuberculosis. The M. abscessus ClpC1 used for experiments was found to contain one residue different from the bacterial strain used for in vitro assays; however, this difference was not present in the NTD. The strain used in SPR contains a G→S mutation near the C terminus at residue 811.
Binding of RUFI (A), ECU (B), and CYMA (C) to wild-type full-length ClpC1 from M. abscessus.
Affinities of binding of cyclopeptides to full-length ClpC1 determined by SPRa
Although overall binding affinities of RUFI and ECU to ClpC1 in equilibrium are similar, association and dissociation rates are very different. Both rates of RUFI are higher, at 1.42 × 106 M−1s−1 and 1.37 × 10−1 s−1, respectively, than those of ECU, at 5.66 × 104 M−1s−1 and 7.01 × 10−3 s−1, respectively (Fig. 8; Table 5). This indicates that RUFI recognizes and binds to ClpC1 much faster and dissociates faster as well, whereas ECU binds slowly and remains bound much longer. The association rate of CYMA seems to be ∼4-fold higher (5.68 × 106 M−1s−1) than that of RUFI, whereas the dissociation rate of CYMA is ∼7-fold lower (2.19 × 10−2 s−1) than that of RUFI and ∼2.7-fold higher than that of ECU to the full-length ClpC1 from M. abscessus. In the case of M. tuberculosis full-length ClpC1, association rates of RUFI and CYMA were very similar, so the overall binding affinity differences mainly came from the ∼13-fold-lower dissociation rate of CYMA than that of RUFI. Collectively, this indicated that the equilibrium binding affinity of CYMA is tighter than those of both RUFI and ECU. The binding affinities for all three compounds were found to be similar for the tested mycobacterial ClpC1 proteins. This is consistent with the observation that these compounds bind the NTD of ClpC1. The lack of significant difference in binding affinity also indicates that the differences in full-length (FL) sequence have only a minimal effect on the binding of ECU, RUFI, or CYMA. However, this does not explain the difference in activity observed in vitro. Therefore, the difference in activity is most likely explained by other cellular factors, such as cellular permeation, enzymatic deactivation, or efflux pumps.
Rufomycin I PK study.Following a single intravenous (i.v.) administration of 5 mg/kg of body weight, RUFI showed behavior in vivo similar to that of ECU (12). Although the formulation {10% EtOH, 40% 10% (2-hydroxypropyl)-β-cyclodextrin [HP-β-CD] solution, 50% polyethylene glycol 400 [PEG 400]} was different from the micelle formulation used for ECU, RUFI was eliminated from the plasma with a mean (n = 3) terminal half-life of 1 h (Fig. 9). This was less than the 3 h observed for ECU. The maximum plasma concentration (C0) was determined to reach 5.1 to 7.4 μg/g, with area under the curve from 0 h to infinity (AUC0–∞) determined to be 1.8 to 2.4 μg/g. This observed difference from ECU can most likely be explained by the difference in formulation, as micellar formulations tend to improve pharmacokinetic (PK) properties of lipophilic compounds. Additionally, there were no clinical abnormalities observed in any of the mice tested.
Mean plasma concentrations of RUFI in mice (n = 3) after a single i.v. dose of RUFI at 5 mg/kg.
This report describes another potent, narrow-spectrum anti-TB cyclic peptide isolated from an actinomycete as a result of an HTS program against M. tuberculosis. This compound, RUF1, also happens to bind to the same molecular target, ClpC1, as ECU but with a different mechanism of action, thereby offering opportunities to further explore the complexities of ClpC1 as a new drug target. As chaperone protease complexes are present in many bacterial families, and an ideal antimycobacterial agent has a narrow spectrum of activity due to the long treatment duration, this study assessed the selectivity of RUFI against several nonmycobacterial species. RUFI was determined to be selective to mycobacteria. With the expectation of targeting MDR and XDR M. tuberculosis, the ability for RUFI to maintain activity against these strains is essential. RUFI maintained activity against all tested monoresistant, MDR, and XDR strains of M. tuberculosis, suggesting that RUFI would be an ideal drug lead for MDR and XDR TB treatment. Based on the reported cytotoxicity of the structurally related anti-TB ohmyungsamycins A and B (31, 32) to several cancer lines, RUFI and ECU were also tested against several cancer cell lines to assess their potential as chemotherapy agents. While ECU lacked significant cytotoxic activity, RUFI demonstrated some toxicity against the tested cancer cell lines while demonstrating no toxicity to Vero cells, indicating an area for further exploration.
Although both ECU and RUFI target ClpC1, their binding and resulting downstream effects are likely to be different, as indicated by WGS of resistant mutants, SPR, and functional assays. This further increases the interest in ClpC1 as a future target for TB drug treatment. The lack of cross-resistance between rECU and rRUFI strains indicates the potential for a broader range of treatment options for resistant and MDR-TB patients. For example, if a patient begins to show resistance to one ClpC1 inhibitor, this resistance may not necessarily preclude the use of a different compound from the same class. The ability of RUFI and ECU to significantly inhibit cellular protein degradation by the ClpC1/P1/P2 complex, as observed in the functional assays, poses additional questions about the downstream effects of these compounds. Although RUFI appears to be more potent (MIC of 0.02 μM) than ECU (MIC of 0.16 μM), the bactericidal effect of ECU (MBC of 0.34 μM) is roughly equal to that of RUFI (MBC of 0.40 μM). In addition, ECU shows activity against nonreplicating cultures (MBC of 1.5 μM), while RUFI apparently lacks this activity. Remarkably, the ECU analogues showed only minimal differences in MIC (33), while the MIC values of the RUF analogues varied greatly with small structural changes, as summarized in Fig. 2. The differences in the biological profiles of these two groups of cyclic peptides suggest that although they bind to the same molecular target, the specific binding patterns and the downstream effects on M. tuberculosis may differ greatly. Studies to observe their effects on ClpC1 and the proteomic consequences of these inhibitors are ongoing.
Finally, and important for further preclinical drug development, RUFI offers a lower-molecular-weight alternative to ECU with potentially less solubility and cellular penetration issues. This is supported by the observed activity of RUFI against M. abscessus versus the lack of activity of ECU, which could not be explained by SPR binding affinity assays. Additionally, the PK behavior of RUFI suggests that this compound may behave similarly to ECU in efficacy-based studies, as both compounds have reasonable serum half-lives and clearance (12). This also leads to the additional application of RUFI as a drug candidate for the treatment of M. abscessus, which often causes skin and soft tissue as well as pulmonary infections (34, 35). Among rapidly growing NTM strains, M. abscessus is considered one of the most virulent and resistant to antimicrobial therapy, thus having limited therapeutic options (26). The present data show the unique potential of RUFI as a drug lead against NTM infections, including M. abscessus infections.
Even though the challenges associated with oral dosing appear to be similar for all investigated cyclic peptides, determining the structural pharmacophore common to RUFI and ECU with the assistance of SAR studies could lead to an improved drug candidate. In addition, efforts to improve the bioavailability of cyclic peptides are gaining traction as their pharmaceutical applications become more apparent (36–41). In summary, this study coupled with others (8–12, 42) demonstrates that ClpC1 is a viable drug target for the treatment of TB.
MICs versus M. tuberculosis.All experiments with M. tuberculosis were conducted within a biosafety level 3 (BSL3) laboratory. The microplate alamarBlue assay (MABA) was used to determine MIC values as previously described (43). Briefly, samples were dissolved in dimethyl sulfoxide (DMSO) prior to addition to the assay plate containing Middlebrook 7H12 medium (Sigma-Aldrich, Darmstadt, Germany). Twofold serial dilution of the compounds was performed. Standard control compounds used were rifampin (RIF), isoniazid (INH), linezolid (LZD), moxifloxacin (MXF), pretomanid (PA-824), bedaquiline (BDQ), capreomycin (CAP), clofazimine (CLO), streptomycin (STR), kanamycin (KM), and cycloserine (CS). All standard compounds were obtained from Sigma-Aldrich except BDQ and PA-824, which were obtained from The Global Alliance for TB Drug Development. Plates were inoculated with M. tuberculosis strain H37Rv (ATCC 27294) and incubated for 7 days at 37°C. Then the redox dye (20 μl of 0.6 mM resazurin dye and 12 μl of 20% Tween 80) was added to each well, and the plates were incubated for an additional 18 to 24 h at 37°C. Final fluorescence was measured at 530 nm excitation and 590 nm emission using either a CLARIOstar (BMG LABTECH, Ortenberg, Germany) or VICTOR X3 (PerkinElmer Inc., Waltham, MA) plate reader. The MIC was defined as the minimum concentration of the compound required to achieve a reduction in fluorescence of 90% relative to untreated bacterial controls.
Cytotoxicity in mammalian cells.Cytotoxicity was tested using the Vero cell (ATCC CRL-81) and J774A.1 macrophage (ATCC TIB-67) lines (44–46). Vero cells were cultured in Eagle's minimum essential medium (MEM) containing 10% fetal bovine serum (FBS) plus penicillin and streptomycin. Vero cells were prepared and washed with 0.25% trypsin-EDTA 1× solution in Hanks' balanced salt solution (HBSS; pH 7.4). J774 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS plus polymyxin B. J774 cells were detached by cell scraper. After verifying the morphology by microscopy and adjusting the density to 3 to 5 × 105 cells/ml in MEM, 100 μl of the cell suspension was incubated with the test compounds at 37°C for 72 h; visual inspection was performed after 24 h. Then 20 μl of 0.6 mM resazurin was added into each well and incubated for 4 h. The fluorescence was measured at excitation/emission wavelengths of 530/590 nm. The concentration of test compound effecting a reduction in fluorescence of 50% relative to untreated cells (IC50) was calculated. Cytotoxicity assessment was repeated using the J774A.1 macrophage line for the interpretation of antimycobacterial activity within the macrophage. RIF and BDQ were included as controls.
Cytotoxicity against selected cancer cell lines.Human melanoma cancer cells (MDA-MB-435), human breast cancer cells (MDA-MB-231), and human ovarian cancer cells (OVCAR3) were purchased from the American Type Culture Collection (Manassas, VA). Each cell line was propagated at 37°C in 5% CO2 in RPMI 1640 medium supplemented with FBS (10%), penicillin (100 U/ml), and streptomycin (100 μg/ml). Cells in log phase were harvested by trypsinization, followed by two washing cycles to remove all traces of enzyme. A total of 5,000 cells were seeded per well of a 96-well clear, flat-bottom plate (Microtest 96; Falcon) and incubated overnight (37°C in 5% CO2). Samples dissolved in DMSO were then diluted and added to the respective wells. The cells were incubated in the presence of test substance for 72 h at 37°C and evaluated for viability with a commercial absorbance assay (CellTiter 96 AQueous One Solution cell proliferation assay; Promega Corp., Madison, WI) that measured viable cells. IC50 values are expressed in micromolar values relative to the solvent (DMSO) control.
MICs against mono-drug-resistant isolates.MICs were assessed against strains of M. tuberculosis H37Rv with mono-drug resistance to RIF (ATCC 35838), INH (ATCC 35822), STR (ATCC 35820), KM (ATCC 35827), CS (ATCC 35826), MXF, and CAP. The rMXF and rCAP strains were isolated in our laboratory and contained mutations within gyrA and tylA, respectively. The assays were carried out with the same protocol as used for susceptible H37Rv strains.
MICs against susceptible, MDR, and XDR clinical strains.Twelve pan-susceptible, eleven MDR, and eleven XDR (total, 34 strains) clinical isolates strains were used. All strains were isolated from patients who were enrolled in a prospective observational cohort study (ClinicalTrials.gov identification number NCT00341601) between 2005 and 2012, and the strains were stored in −70°C deep freezer at the International Tuberculosis Research Center (ITRC; Masan, South Korea) before use. All experiments utilizing M. tuberculosis were performed in a BSL3 laboratory in ITRC. Phenotypic drug susceptibility testing was performed by using the Bactec MGIT 960 system according to the manufacturer's recommendations (47) with the following final drug concentrations: 0.1 μg/ml for INH, 1.0 μg/ml for RIF, 5.0 μg/ml for ethambutol (EMB), 1.0 μg/ml for STR, 2.5 μg/ml for KM, 2.5 μg/ml for CAP, 2.0 μg/ml for ofloxacin (OFX), and 0.25 μg/ml for MXF. For the detection of genotypes, all isolates were subjected to whole-genome sequencing; a paired-end sequencing library was constructed with a 500-bp insert size using the NexTera sample preparation kit (Illumina, San Diego, CA) for the Illumina-HiSeq platform, and genotypic drug resistance characteristics for INH and RIF were identified using PhyResSE version 1.0 (bioinf.fz-borstel, Germany).
When performing the susceptibility testing with RUFI, INH and RIF were included to confirm the validity of the test results. The drug susceptibility testing was performed by using the Bactec MGIT 960 system according to the manufacturer's recommendations (47) with the following final drug concentrations: 0.1 μg/ml for INH, 1.0 μg/ml for RIF, and 0.03, 0.06, 0.125, 0.25, 0.5, and 1.0 μg/ml for RUFI.
MICs against nonmycobacteria.MICs, defined as the lowest concentration resulting in ≥90% inhibition of bacterial growth relative to bacterial control against the nonmycobacterial strains Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 29213), were determined by measuring optical density at a wavelength of 570 nm (OD570) after 16 h of incubation at 37°C in 2.2% Mueller-Hinton II broth (CAMH; Becton, Dickinson, Sparks, MD). Standard compounds used were ampicillin (AMP) and gentamicin (GEN). The MIC against Candida albicans (ATCC 10231) was determined after 48 h of incubation at 37°C by measuring OD570 in 1% Cellgro RPMI 1640 medium (Mediatech Inc., Manassas, VA) supplemented with 1.8% d-(+)-dextrose (ICN Biomedicals, Aurora, OH) and 3.5% morpholinopropanesulfonic acid (MOPS) (Acros, NJ). Amphotericin B (AmphB) and ketoconazole (KETO) were used as standard compounds.
MICs against nontuberculous mycobacteria.MICs against M. abscessus (ATCC 19977) and M. smegmatis (ATCC 700084) were determined after 72 h of incubation at 37°C followed by addition of 0.6 mM resazurin and 20% Tween 80 and an additional 4 h of incubation. M. chelonae (ATCC 35752) was incubated for 72 h, and M. marinum (ATCC 927) for 120 h, both at 30°C. The MIC against M. bovis (ATCC 35734) was determined with the MABA protocol. M. kansasii (ATCC 12478) and M. avium (ATCC 15769) were incubated at 37°C for 6 and 7 days, respectively. All strains were cultured using Middlebrook 7H9 medium supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC).
MICs against genetically diverse M. tuberculosis.MIC values were determined by the MABA protocol against a panel of M. tuberculosis isolates that were collected from around the world and grouped into six major single nucleotide polymorphism (SNP) clusters, five of which were tested (48). Tested isolates were X001354 from the East African Indian lineage, X004439 and X004244 from the East Asian lineage, and X005282 and X005319 from the Euro-American lineage. X001354, representing the Indo-Oceanic lineage, was not tested.
MBC against M. tuberculosis and M. abscessus.The minimum bactericidal concentration (MBC) against M. tuberculosis was determined for RUFI and the standard compound RIF following the MABA protocol, with each compound run in triplicate. Two sets of plates were run in parallel; after the 7-day incubation period, one plate was analyzed following MABA protocol, with the addition of resazurin and Tween 80, while no dye was added to the second plate. Instead, the contents of the inoculated test wells were collected into 1.5-ml tubes, washed with phosphate-buffered saline (PBS), and then plated on 7H11 agar. Plates were incubated at 37°C for 2 to 3 weeks before CFU determination. The MBC was defined as the lowest concentration reducing CFU by 99% relative to the time-zero inoculum.
The MBC against M. abscessus was determined for RUFI and the standard compound BDQ by following the MABA protocol, with a 72-h incubation in 7H9 medium supplemented with OADC. Parallel plates were processed as described for M. tuberculosis. The plates were incubated at 37°C for 5 days before CFU determination. The MBC was defined as the lowest concentration reducing CFU by 99% relative to the time-zero inoculum.
Activity in macrophage culture.Inhibition of M. tuberculosis Erdman (ATCC 35801) in macrophage culture was assessed as previously described (12, 45, 49). Briefly, J774A.1 cells were grown, adjusted to a concentration of 1 × 105 to 3 × 105/ml, and then distributed to 13-mm coverslips in 24-well plates to facilitate the washing out of nonphagocytosed M. tuberculosis. Cells were then incubated at 37°C with 5% CO2 overnight prior to infection with M. tuberculosis. The M. tuberculosis inoculum was prepared at a concentration of 1 × 105 to 3 × 105 CFU/ml in DMEM and transferred to a fresh 24-well plate. J774A.1 coverslips were transferred to the 24-well plate containing the M. tuberculosis, and the plate was incubated for 1 h at 37°C with 5% CO2. Next bacteria were washed from the cell coverslips with HBSS, and then fresh DMEM was added to the wells. After the plates were incubated overnight to allow phagocytosis, they were washed three times with warm HBSS and transferred to a fresh 24-well plate containing 1 ml of diluted compound. RUFI was tested at 0.019, 0.098, and 0.48 μM versus the control compound, RIF, at 0.024, 0.12, and 0.61 μM. The plates were then incubated for 6 days at 37°C and 5% CO2. All experiments were run in triplicate. For the time-zero (T0) untreated control and the other cultures after 6 days of incubation, macrophages were removed, subjected to lysis with 200 μl of 0.25% sodium dodecyl sulfate (SDS), and incubated at 37°C for 10 min. Fresh medium was then added, and samples were sonicated, diluted, plated on 7H11 agar, and incubated at 37°C. The CFU were determined following 2 to 3 weeks of incubation.
Intracellular survival assay in BMDMs.Bone marrow-derived macrophages (BMDMs) were isolated from the femurs and tibias of C57BL/6 mice (5 to 6 weeks old) and differentiated by culturing for 4 days in DMEM containing 10% (vol/vol) FBS and 25 ng/ml of macrophage colony-stimulating factor (M-CSF; R&D, Minneapolis, MN). The final densities of differentiated BMDMs were 1 × 105 to 3 × 105 per well in 48-well plates. Cells were infected for 2 h with M. abscessus at a multiplicity of infection (MOI) of 5. Next cells were washed with PBS and incubated in DMEM containing 50 μg/ml of gentamicin (Sigma-Aldrich). RUFI and clarithromycin (CLR) were added to the media to achieve final concentrations of 5 and 10 μM, respectively, and then the cultures were incubated for 2 days. The supernatant was removed, and cells were lysed in sterile distilled water for 30 min at 37°C. Cell lysates were serially diluted, plated on 7H10 agar, and incubated at 37°C for 2 to 3 days for enumeration of CFU.
Selection of rufomycin-resistant strains of M. tuberculosis.M. tuberculosis mutants resistant to RUFI were generated by plating H37Rv on 7H11 agar plates containing 1 μg/ml and 5 μg/ml of RUFI. Plates were prepared by adding the appropriate volume of RUFI in DMSO to molten 7H11 medium. A total of 4 × 109 CFU/ml of M. tuberculosis were plated on the agar plates and incubated for 3 to 4 weeks at 37°C. Colonies were then streaked on 7H11 agar plates containing 5 μg/ml and 10 μg/ml of RUFI and incubated for 3 to 4 weeks at 37°C. This process was repeated for 10 μg/ml and 20 μg/ml and then 20 μg/ml and 40 μg/ml. Colonies that still grew at the highest concentration of RUFI were picked for inoculation in liquid 7H9 medium containing 0.001× MIC of RUFI and incubated at 37°C for 1 to 2 weeks. The harvested cells were filtered using an 8.0-μm sterile filter, washed with PBS, and stored at −80°C. The MIC determination against these strains was done using the MABA protocol except that plates were incubated for 10 days prior to the addition of resazurin and Tween 80.
Whole-genome sequencing of rufomycin-resistant M. tuberculosis.Genomic DNAs of the rRUFI M. tuberculosis strains (#28, 31, 35, 41, 50, 51, and 53) were isolated as described previously (50). Genomic DNA extracted from each of these M. tuberculosis strains and from wild-type H37Rv was sequenced on a HiSeq 2000 instrument (Illumina, San Diego, CA). The raw sequence data were imported into the CLC Genomics Workbench software package (Qiagen, Redwood City, CA; v5.5) as paired reads. Stringent trimming was performed (0.01 quality trimming threshold with no degeneracies allowed), and all sequences shorter than 100 bases after trimmings were discarded. After trimming, approximately 5 to 11.5 million reads were recovered from each sample. These reads were mapped to the reference genome of M. tuberculosis H37Rv (GenBank accession no. NC_000962) using default assembly parameters (0.5 length fraction and 0.8 similarity fraction) implemented within CLC Genomics Workbench. The variants relative to the reference genome, including single nucleotide variants as well as insertions and deletions, were detected using the probabilistic variant detection routine within CLC Genomics Workbench. The parameters included a minimum coverage of 50× and a minimum variant probability of 50%. The reads from each strain were inspected with IGV software (Broad Institute) to identify any mutations missed due to coverage gaps and low read scores. PCR, Sanger DNA sequencing, and Sequencher (Gene Codes, Ann Arbor, MI) were used to confirm nonsynonymous variants.
Mycobacterium clpC1 expression and purification for surface plasmon resonance (SPR).The coding sequence of clpC1 (Rv3596c) was amplified by PCR from genomic DNA of the wild-type M. tuberculosis strain H37Rv and ligated to create a recombinant pET-30a(+) plasmid (Novagen EMD Millipore, Darmstadt, Germany) with C-terminal His tag. The wild-type clpC1 PCR fragment was cloned into the NdeI and HindIII restriction enzyme (RE) sites of pET-30a(+). M. abscessus and M. chelonae ClpC1 plasmids were obtained from Genscript (Piscataway, NJ) using M. tuberculosis clpC1 as a DNA template for mutagenesis. All recombinant plasmids were transformed into BL21-Gold (DE3) expression-competent E. coli (Agilent Technologies, Santa Clara, CA) for isopropyl-β-d-thiogalactopyranoside-induced protein expression at 37°C. Plasmid sequences were confirmed with Sanger DNA sequencing performed by the Research Resource Center at University of Illinois at Chicago (UIC). Frozen cells were lysed by a 30-min incubation on ice with lysozyme (1 mg/ml), benzonase nuclease (3 U/ml; Novagen), and a complete protease inhibitor tablet (EDTA free; Roche). After centrifugation, the C-terminal His-tagged protein was purified from the cell lysate with a nickel-nitrilotriacetic acid (Ni-NTA) spin column (Qiagen) preequilibrated with buffer A (20 mM phosphate [pH 8.0], 300 mM NaCl). The column was washed with buffer A plus 20 mM imidazole, and the protein was eluted in buffer A with 500 mM imidazole. Purity was evaluated by SDS-PAGE, and the protein was shown to migrate according to an approximate size of 95 kDa. The protein concentration was determined at A280 using the calculated extinction coefficient (ε280 = 35,870 M−1 cm−1), and then the buffer was exchanged (Zebra spin desalting column) into PBS with 15% glycerol and the protein was stored at −80°C.
Analysis of cyclopeptide binding affinity to mycobacterial ClpC1 by SPR.Each ClpC1 enzyme was immobilized on a CM5 sensor chip using standard amine coupling at 25°C with running buffer PBS-P (20 mM phosphate, 137 mM NaCl, 27 mM KCl, 0.05% surfactant P-20 [pH 7.4]) using a Biacore T200 instrument (GE Healthcare Bio-Sciences, Pittsburgh, PA). Unmodified blank surface was used on flow channel 1 as a control. ClpC1 enzymes were diluted with 10 mM sodium acetate (pH 4.0) to 100 μg/ml and immobilized to flow channels 2, 3, and 4 after sensor surface activation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxy succinimide (NHS) followed by ethanolamine blocking on unoccupied surface area. Binding of RUFI, ECU, and CYMA to wild-type full-length ClpC1 from M. tuberculosis, M. abscessus, and M. chelonae were monitored in SPR binding buffer (PBS-P, 0.5 mM Tris(2-carboxyethyl)phosphine hydrochloride [TCEP], 2% DMSO) at 25°C and a flow rate of 30 μl/min. A series of increasing concentrations of the compounds were run over the surface of the immobilized full-length ClpC1 proteins. Data were double referenced with blank-channel and zero-concentration responses.
Kinetic association rate (ka) and dissociation rate (kd) constants were determined by fitting globally to the 1:1 Langmuir model embedded in the Biacore T200 evaluation software (v3.0). The KD (equilibrium dissociation constant) values were calculated from the determined rate constants (KD = kd/ka). Response units at each concentration were also measured during the equilibrium phase for steady-state affinity fittings, and the KD values were determined by fitting the data to a single rectangular hyperbolic curve equation (equation 1), where y is the response, ymax is the maximum response, and x is the compound concentration.y=ymax×xKD+x (1)
Purification of recombinant ClpC1, ClpP1, and ClpP2 for functional assays.Recombinant ClpC1 was expressed in E. coli Rosetta2 (DE3) (Novagen). Recombinant ClpP1 and ClpP2 were expressed in E. coli BL21(DE3) (Novagen). Purification of all proteins was carried out by Ni-tris-carboxymethyl ethylene diamine (TED) chromatography (Macherey-Nagel, Oensingen, Switzerland) following the user instruction manual. Briefly, E. coli cells were harvested by centrifugation (6,000 × g, 30 min, and 4°C) and broken for 10 min on ice using a sonicator (Branson, Emerson Industrial Automation, Danbury, CT). The soluble fraction was collected after centrifugation (12,000 × g, 30 min, and 4°C) and then passed through a 0.2-μm syringe filter for complete clearance of aggregates. After purification, the purity of the proteins was monitored by electrophoresis using SDS-PAGE. The protein concentration was determined using the bicinchoninic acid (BCA) protein assay reagent (Pierce Chemical Co., Dallas, TX) with standard bovine serum albumin.
Measurement of ATPase activity of recombinant ClpC1.The measurement of ClpC1 ATPase activity was carried out by the BIOMOL Green modified malachite green assay (51). The reaction mixture was prepared in assay buffer (100 mM Tris, 200 mM KCl, 8 mM MgCl2 [pH 7.5]), and the final volume of the reaction mixture was 50 μl. In the reaction mixture, the final concentrations of ClpC1 and ATP were 1 μM and 100 μM, respectively. For the measurement of the ATPase activity by treatment with ECU and RUFI, each compound, dissolved in DMSO, was added at 0.1, 1.0, and 10 μM as the final concentrations in each well of a transparent 96-well plate. To avoid any effect of protein denaturation by DMSO, 1 μl of compound solution was added to the reaction mixture. The reaction mixture was incubated at 37°C for 1 h, and then 50 μl of BIOMOL Green solution (Enzo Life Science, Farmingdale, NY) was added to the reaction mixture. After incubation for 10 min at room temperature, the OD of the reaction mixture was measured at 620 nm on an Infinite 200 PRO (Tecan, Switzerland). The concentration of free phosphate released from ATP by ClpC1 was calculated using a standard curve according to the concentration of free phosphate.
Measurement of proteolytic activity.Proteolytic activity of ClpC1, ClpP1, and ClpP2 was monitored by degradation of fluorescein isothiocyanate (FITC)-casein (Sigma-Aldrich) as the substrate (52). The reaction mixture was prepared in buffer used in the assay for ClpC1 ATPase activity, and the final volume of the reaction mixture was 100 μl. In the reaction mixture, the final concentrations of ClpC1, ClpP1, and ClpP2 were 1, 2, and 2 μM, respectively. In addition, 100 μM ATP and 1 μM FITC-casein were added to each reaction mixture. For the measurement of the degradation of FITC-casein by the ClpC1, ClpP1, and ClpP2 complexes in the presence of ECU and RUFI, each compound, dissolved in DMSO, was added at 0.1, 1.0, and 10 μM in each well of the black 96-well plates with a transparent bottom. To avoid protein denaturation by DMSO, only 2 μl of the compound solution was added to the reaction mixture. The reaction mixture was incubated at 37°C for 1 h, and then the fluorescence level of FITC-casein was measured at 485-nm excitation and 535-nm emission on an Infinite 200 PRO instrument (Tecan, Switzerland).
Classification of Streptomyces sp. MJM3502 by 16S rRNA gene sequence.Streptomyces sp. MJM3502 genomic DNA was extracted following the protocol of CoreBio (Republic of Korea) genomic DNA isolation kit. The 16S rRNA gene was amplified and sequenced by using the universal primers 27F (forward; 5′-AGAGTTTGATCATGGCTCAG-3′) and 1492R (reverse; 5′-AAGGAGGTGATCCARCCGCA-3′. The sequence was aligned with the closest Streptomyces strains in BLAST using CLUSTAL X (53), and the phylogenetic tree was constructed by the neighbor-joining method (54) using MEGA4.0 software (55). The topology of the phylogenetic tree was calculated by 1,000 bootstrap replications (56).
Mouse pharmacokinetic study.Healthy female BALB/c mice were administered a single dose (5 mg/kg) of RUFI intravenously (i.v.; tail vein). RUFI was formulated using 10% ethanol (EtOH), 40% 10% (2-hydroxypropyl)-β-cyclodextrin (HP-β-CD) solution, and 50% PEG 400. RUFI was first dissolved in 0.102 ml of EtOH, vortexed and sonicated until clear, and then diluted with 0.508 ml of PEG 400 with additional vortex treatment and sonication until clear. Finally, 0.406 ml of 10% HP-β-CD was added to the solution. Then 20 μl of this formulation was pipetted into a 10-ml volumetric flask, and methanol (MeOH) was added to a final concentration of 1 mg/ml. The accuracy of the formulation concentration was verified via high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) (AB Sciex 6500+; SCIEX, Framingham, MA) by electrospray using multiple reaction monitoring (MRM; 1065.63/1047.70) in positive ion mode. LC was performed with a Kinetex EVO C18 column (50 mm by 3.00 mm [inside diameter], 2.6-μm particle size, 100-Å pore size) using a gradient of 0.1% formic acid (FA) in water–0.1% FA in acetonitrile (ACN) (25% ACN to 95% ACN in 1.5 min) at a flow rate of 0.8 ml/min.
Blood samples (10 μl) were collected via saphenous vein puncture at 5, 15, and 30 min and 1, 2, 4, 8, and 25 h. To each of the blood samples collected, 2.0 μl of MeOH was added followed by 200 μl of a solution of 5 ng/ml of terfenadine (internal standard) in MeOH. Samples were then vortexed for 1 min and centrifuged at 4,000 rpm for 15 min. The supernatant was collected and diluted with water, 2× (vol/vol) for HPLC-MS/MS analysis, as described above for the formulation determination. A standard curve was determined by using RUFI spiked into untreated mouse plasma.
We thank Chambers C. Hughes of The Scripps Institution of Oceanography (SIO), University of California San Diego, for supplying cyclomarin A. We also thank Joanna E. Burdette from the University of Illinois at Chicago (UIC) for testing the compounds against cancer cell lines. We also acknowledge the pharmacokinetic work of BioDuro Inc. and the genomic analysis work of the Research Resources Center (RRC) at UIC.
This work was supported by the following: grant T32AT007533 from NCCIH/NIH (supporting M.P.C.), grant R21 AI093919-02 from NIIAD/NIH (to B.U.J., principal investigator [PI]), the Next-Generation BioGreen 21 Program (no. PJ01328403 to S.C. as PI and no. PJ01319101 to J.-W.S. as PI), the Rural Development Administration, Republic of Korea, and the National Research Foundation of Korea (NRF) grant funded by the Korean Government (MSIP) (no. 2017R1A5A2015385 to S.P., J.H.C., and E.-K.J.). We also acknowledge the construction of the UIC CSB, the acquisition of the Jasco-815 spectropolarimeter, and the purchase of the 900-MHz nuclear magnetic resonance (NMR) spectrometer funded by grant P41 GM068944 from NIGMS/NIH awarded to Peter Gettins as PI.
Received 19 October 2018.
Returned for modification 7 November 2018.
Accepted 14 December 2018.
Accepted manuscript posted online 2 January 2019.
Copyright © 2019 Choules et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
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. 2015. Probing the physicochemical boundaries of cell permeability and oral bioavailability in lipophilic macrocycles inspired by natural products. J Med Chem 58:4581–4589. doi:10.1021/acs.jmedchem.5b00128.
Hoang HN,
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. 2014. Improving on nature: making a cyclic heptapeptide orally bioavailable. Angew Chem Int Ed Engl 53:12059–12063. doi:10.1002/anie.201405364.
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. 2010. Functional characterization of the cyclomarin/cyclomarazine prenyltransferase CymD directs the biosynthesis of unnatural cyclic peptides. J Nat Prod 73:373–377. doi:10.1021/np9006876.
Lee HS,
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. 2015. Microplate alamar blue assay (MABA) and low oxygen recovery assay (LORA) for Mycobacterium tuberculosis. Methods Mol Biol 1285:281–292. doi:10.1007/978-1-4939-2450-9_17.
DeGroote MA,
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. 2012. Comprehensive analysis of methods used for the evaluation of compounds against Mycobacterium tuberculosis. Tuberculosis (Edinb) 92:453–488. doi:10.1016/j.tube.2012.07.003.
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. 2010. Targeting Mycobacterium protein tyrosine phosphatase B for antituberculosis agents. Proc Natl Acad Sci U S A 107:4573–4578. doi:10.1073/pnas.0909133107.
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. 2007. Multicenter laboratory evaluation of the MB/BacT Mycobacterium detection system and the BACTEC MGIT 960 system in comparison with the BACTEC 460TB system for susceptibility testing of Mycobacterium tuberculosis. J Clin Microbiol 45:1766–1770. doi:10.1128/JCM.02162-06.
Filliol I,
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. 2006. Global phylogeny of Mycobacterium tuberculosis based on single nucleotide polymorphism (SNP) analysis: insights into tuberculosis evolution, phylogenetic accuracy of other DNA fingerprinting systems, and recommendations for a minimal standard SNPs. J Bacteriol 188:759–772. doi:10.1128/JB.188.2.759-772.2006.
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. 2005. In vitro and in vivo activities of macrolide derivatives against Mycobacterium tuberculosis. Antimicrob Agents Chemother 49:1447–1454. doi:10.1128/AAC.49.4.1447-1454.2005.
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Antimicrobial Agents and Chemotherapy Feb 2019, 63 (3) e02204-18; DOI: 10.1128/AAC.02204-18
You are going to email the following Rufomycin Targets ClpC1 Proteolysis in Mycobacterium tuberculosis and M. abscessus
ClpC1
Mycobacterium abscessus
cyclic peptide
rufomycin


People often paint a dark picture of you. But these songs seem like your way of casting those feelings away. To me that's ultimately optimistic.
Well, I find the whole process of writing and recording a positive thing to do. It can't be anything else. If you want to use that language, it is an optimistic thing to do. I know I've written some heavy going songs in my days. And I apologize to everyone for that. But I've never considered them depressing.
Do you remember when music touched you in a way where you knew it would always be part of your life?
I'm still surprised that it is. It was never what I was going to do. It was just an accident. I wanted to be a painter. But through a series of accidents I ended up being a musician.
We talked earlier about how for you, it's always about moving on to the next project.  What were you working on today?
Well, I'm writing a novel. And today I killed off the central character. I'm quite pleased about that. It was a big day for him. (laughs) He is now officially dead, which means I'm nearly finished the book.  So that's been quite exciting.
Everything inspires. Other peoples' art inspires me. Everything that I see. But in the end, I work. I sit down, put my head down and I work. And inspiration doesn't really have that much to do with it. It's more about writing one word after the next.
Nick Cave And The Bad Seeds perform at the WaMu Theater at Madison Square Garden in NYC on Oct. 4. For more information, visit nickcaveandthebadseeds.com or myspace.com/nickcaveandthebadseeds.


Dear friends, on the weekend of Friday 5th of April until Sunday the 7th, the MEBC have been invited on a weekend retreat to Adhisthana. The main purpose of the weekend will be for the MEBC to ritually place a small Buddha figure into the main wall behind the shine at Adhisthana, where all Triratna centres are also represented by a small figure. The full cost of the weekend is £120 (including all food & refreshments) Katrina shall be taking booking as of the 15/01/19. Please note that spaces are limited to the first 30 people, therefore if you do wish to attend this retreat please inform Katrina as soon as possible, to avoid disappointment.


For the occasion of Amsterdam Denim Days 2016 an annual event for fashion forward denim lovers, starting this Monday! I thought I show you my latest denim find in town 'The Blauw kitchen' at de Pijp area, a beautiful addition to the Scotch & Soda store chain.
'The Blauw kitchen' is a beautiful small denim concept store with an interior based on denim, and Japanese denim technics while the white tiles refer to the kitchen and denim lab where can be experimented with different colouring technics.


text:
A solar mirror contains a substrate with a reflective layer for reflecting the solar energy, and in most cases an interference layer.  This may be a planar mirror or parabolic arrays of solar mirrors used to achieve a substantially concentrated reflection factor for solar energy systems.

See article "Heliostat" for more information on solar mirrors used for terrestrial energy.

Components

Glass or metal substrate

The substrate is the mechanical layer which holds the mirror in shape.

Glass may also be used as a protective layer to protect the other layers from abrasion and corrosion.  Although glass is brittle, it is a good material for this purpose, because it is highly transparent (low optical losses), resistant to ultraviolet light (UV), fairly hard (abrasion resistant), chemically inert, and fairly easy to clean.  It is composed of a float glass with high optical transmission characteristics in the visible and infrared ranges, and is configured to transmit visible light and infrared radiation.  The top surface, known as the "first surface", will reflect some of the incident solar energy, due to the reflection coefficient caused by its index of refraction being higher than air.  Most of the solar energy is transmitted through the glass substrate to the lower layers of the mirror, possibly with some refraction, depending on the angle of incidence as light enters the mirror.

Metal substrates ("Metal Mirror Reflectors") may also be used in solar reflectors.  NASA Glenn Research Center, for example, used a mirror comprising a reflective  aluminum surface on a metallic honeycomb as a prototype reflector unit for a proposed power system for the International Space Station.  One technology uses aluminum composite reflector panels, achieving over 93% reflectivity and coated with a speciality coating for surface protection.  Metal reflectors offer some advantages over glass reflectors, as they are lightweight and stronger than glass and relatively inexpensive. The ability to retain parabolic shape in reflectors is another advantage, and normally the subframe requirements are reduced by more than 300%. The top surface reflection coating allows for better efficiency.

Reflective layer
The reflective layer is designed to reflect the maximum amount of solar energy incident upon it, back through the glass substrate.  The layer comprises a highly reflective thin metal film, usually either silver or aluminum, but occasionally other metals.  Because of sensitivity to abrasion and corrosion, the metal layer is usually protected by the (glass) substrate on top, and the bottom may be covered with a protective coating, such as a copper layer and varnish.

Despite the use of aluminum in generic mirrors, aluminum is not always used as the reflective layer for a solar mirror.  The use of silver as the reflective layer is claimed to lead to higher efficiency levels, because it is the most reflective metal.  This is because of aluminum's reflection factor in the UV region of the spectrum. Locating the aluminum layer on the first surface exposes it to weathering, which reduces the mirror's resistance to corrosion and makes it more susceptible to abrasion.  Adding a protective layer to the aluminum would reduce its reflectivity.

Interference layer
An interference layer may be located on the first surface of the glass substrate.  It can be used to tailor the reflectance.  It may also be designed for diffuse reflectance of near-ultraviolet radiation, in order to prevent it from passing through the glass substrate. This substantially enhances the overall reflection of near-ultraviolet radiation from the mirror. The interference layer may be made of several materials, depending on the desired refractive index, such as  titanium dioxide.

Passive mirror cooling applications
The use of solar mirrors as a form of passive daytime radiative cooling for solar radiation management has been proposed to address local temperature increases as well as to decrease global warming. Propositions have focused on the usage of solar mirrors both on the Earth's surface and in space.

Terrestrial applications

Passive mirror cooling systems reduce temperatures by reflecting solar radiation while shielding the base of the mirrors from heat penetration. The effectiveness of such systems may be reduced with the accumulation of dust on mirrors, with maximum dust accumulation reducing mirror effectiveness by 63%. However, mirrors may be "self-cleaned" by rain (reducing the soiling rate to 18.6%) or cleaned by humans.

On a local scale, passive mirror cooling systems have been implemented to lower the energy consumption used to cool residential and commercial buildings and thus offset the need for air-conditioning. When passive mirrored surfaces are placed on roofs, they have been shown to reduce electricity consumption and costs for cooling, with one case study reducing costs by 15%.

While the use of solar mirrors as a form of solar radiation management on a global scale has been proposed, more data and funding is required. Increasing awareness of passive radiative cooling's potential to lower costs as well as its role in reducing solar radiation may increase applications. Researchers who support passive mirror cooling applications on a mass scale, such as Ye Tao of MEER, argue that carbon dioxide removal alone will not work fast enough to prevent global temperature increases from surpassing life-threatening levels.

Space-based applications

Solar thermal applications
The intensity of solar thermal energy from solar radiation at the surface of the earth is about , of area normal to the direction of the sun, under clear-sky conditions.  When solar energy is unconcentrated, the maximum collector temperature is about .  This is useful for space heating and heating water.  For higher temperature applications, such as cooking, or supplying a heat engine or turbine-electrical generator, this energy must be concentrated.

Terrestrial applications
Solar thermal systems have been constructed to produce concentrated solar power (CSP), for generating electricity. The large Sandia Lab solar power tower uses a Stirling engine heated by a solar mirror concentrator.  Another configuration is the trough system.

Space power application

"Solar dynamic" energy systems have been proposed for various spacecraft applications, including solar power satellites, where a reflector focuses sunlight on to a heat engine such as the Brayton cycle type.

Photovoltaic augmentation

Photovoltaic cells (PV) which can convert solar radiation directly into electricity are quite expensive per unit area.  Some types of PV cell, e.g. gallium arsenide, if cooled, are capable of converting efficiently up to 1,000 times as much radiation as is normally provided by simple exposure to direct sunlight.

In tests done by Sewang Yoon and Vahan Garboushian, for Amonix Corp. silicon solar cell conversion efficiency is shown to increase at higher levels of concentration, proportional to the logarithm of the concentration, provided external cooling is available  to the photocells.  Similarly, higher efficiency multijunction cells also improve in performance with high concentration.

Terrestrial application

To date no large scale testing has been performed on this concept.  Presumably this is because the increased cost of the reflectors and cooling generally is not economically justified.

Solar power satellite application

Theoretically, for space-based solar power satellite designs, solar mirrors could reduce PV cell costs and launch costs since they are expected to be both lighter and cheaper than equivalent large areas of PV cells.  Several options were studied by Boeing corporation.  In their Fig. 4. captioned "Architecture 4. GEO Harris Wheel", the authors describe a system of solar mirrors used to augment the power of some nearby solar collectors, from which the power is then transmitted to receiver stations on earth.

Space reflectors for night illumination

Another advanced space concept proposal is the notion of space reflectors which reflect sunlight on to small spots on the night side of the Earth to provide night time illumination.  An early proponent of this concept was Dr. Krafft Arnold Ehricke, who wrote about systems called "Lunetta", "Soletta", "Biosoletta" and "Powersoletta".

A preliminary series of experiments called Znamya ("Banner") was performed by Russia, using solar sail prototypes that had been repurposed as mirrors.  Znamya-1 was a ground test.  Znamya-2 was launched aboard the Progress M-15 resupply mission to the Mir space station  on 27 October 1992.   After undocked from Mir, the Progress deployed the reflector. This mission was successful in that the mirror deployed, although it did not illuminate the Earth. The next flight Znamya-2.5 failed. Znamya-3 never flew.

In 2018, Chengdu, China, announced plans to place three solar reflectors in orbit around the Earth in hopes of reducing the amount of electricity required to power streetlights. Skepticism has been voiced regarding the technological feasibility of the plan.

See also

 Heliostat
 Parabolic trough
 Solar thermal collector
 Photovoltaics
 Passive solar
 Solar tracker
 Sun gun

References

Mirrors
Solar thermal energy
Solar-powered devices
Photovoltaics

redpajama_set_name: RedPajamaWikipedia


text:
Q: How to suppress view button Push Notifications? In the apple documentation, they show a notification where there's no view button, just an "OK" button.
How could I do this in my app.
does this have to be implemented in the payload code, or in the local app?

any help is appreciated, thanks!

A: nevermind, I found it has to do with the "body" element of the message:
Here's an example
{"aps":
     {"alert":{"body":"You got a new message!",
     /* You can change "action-loc-key" to what you want the 'action' button
       to say. (In this case, nothing) */
     "action-loc-key":null},
     "badge":0,
     "sound":"beep.wav"},
"acme1":"bar",
"acme2":42
}

redpajama_set_name: RedPajamaStackExchange


text:
Report from Tennessee Department of Education shows Rutherford County Schools outperformed state in all grades for English language arts, math, science and social studies
Sep 30, 2021 at 10:23 am by WGNS
Last school year was unlike any other and, yet Rutherford County educators rose to the challenge of educating nearly 48,000 students amid a worldwide pandemic.
An impressive 41 of 46 schools in the county earned a level three, four or five distinction according to the newly announced student growth data released by the Tennessee Department of Education.
Thirty-five of those schools — or 76 percent— earned a level four or five, both of which are above expectations, according to the TDOE rankings. And 30 of those 35 schools "earned the highest possible" distinction available — which is a level five.
"We had over 40 percent of the student population out for distance learning, which is unprecedented and created a lot of challenges, but the growth we're seeing in this report is a testimony of our teachers and educators to empower our students to grasp tomorrow's opportunities," Director of Schools Bill Spurlock said. "Even as the pandemic continues, we still need to provide that daily, and our teachers are making those opportunities a reality."
Dr. Jimmy Sullivan, assistant superintendent of curriculum and instruction, also noted a collective rise to the challenges of educating more than 48,000 students.
"Growth must occur for all students, period," Sullivan said of the mantra he and his district-level instruction department continually remind educators throughout the school year.
"What I want us to be proud of is that compared to students across the state of Tennessee, our students outperformed expectations and other school districts second to none. They — our teachers, students and our parents — absolutely had that collective effort to show tremendous growth during an extremely trying year. For us to have 30 of our 46 schools outperform everybody across the state … is a huge deal for them."
Student growth is measured year over year by comparing student performance to their own prior performance and is typically not related to a student's demographics.
Scroll down for more...
Growth is critical to ensuring a student's future academic success.
"Yes, we want as many kids as possible to be on grade level," Sullivan said, "but what's more important for us and what we talk to our teachers about all the time is making sure that our students grow every single year they're with us in Rutherford County. We may not be able to control how a student enters us — whether that's in kindergarten or seventh grade or high school — but what we can control is how much they grow while they're with us."
Sullivan reemphasized, "There's no excuses for our kids not to grow."
By comparison to growth data, achievement scores measure a student's performance at one single point in time and compares that performance to a standard that is highly correlated with their demographic.
Achievement scores are critical to a student's post-secondary opportunities.
In addition to the overwhelming growth numbers, Rutherford County outperformed the state in all grades for English language arts, math, science and social studies with a graduation rate of 95.3 percent and an overall districtwide attendance rate of 96 percent.
"We've never performed like that before — growth-wise," Sullivan said, "and that truly goes to our teachers because they're the ones that are heavily ensuring kids leave us better off than they were. And that's ultimately what growth is — are kids in a better place academically than they were when they entered our schools that year? And compared to peers across the state that year, yeah."
"Obviously, we have some challenges as far as achievement is concerned," Spurlock concluded, "but at the end of the day it is all about growth and it is about each individual student."
Scroll down for the list of level five schools...
The following is an alphabetical list of all 30 level five schools, which indicates significant evidence that the school's students made more growth than expected.
- Blackman Elementary
- Blackman High
- Blackman Middle
- Brown's Chapel Elementary
- Buchanan Elementary
- Cedar Grove Elementary
- Central Magnet
- Eagleville
- Holloway High
- Homer Pittard Campus
- John Colemon
- LaVergne High
- Lavergne Lake Elementary
- LaVergne Middle
- Oakland High
- Rock Springs Middle
- Rockvale High
- Rockvale Middle
- Rocky Fork Middle
- Siegel MiddleSmyrna Elementary
- Smyrna High
- Smyrna Middle
- Stewarts Creek Elementary
- Stewarts Creek High
- Stewarts Creek Middle
- Stewartsboro Elementary
- Thurman Francis Arts Academy
- Whitworth-Buchanan Middle
- Wilson Elementary
The following is an alphabetical list of the nine Rewards Schools from Rutherford County.
A reward school is one that maintained the highest performance level available across multiple accountability measures,
per the Tennessee Department of Education.
- McFadden School of Excellence
Tags: English language arts math Rutherford County Schools science social studies Tennessee Department of Education TN
Rutherford County Records a 2.6% Unemployment Rate
Outstanding Fairs were Honored at 100th Annual Convention in Murfreesboro

redpajama_set_name: RedPajamaCommonCrawl


text:
Democracy Without Borders Kenya
Campaign for a UN Parliamentary Assembly
Campaign for a UN World Citizens' Initiative
Global Voting Platform
Global Week of Action for a World Parliament
Other issues we care about
A renewed world organization for the 21st century
United Humans
A voice for global citizens
Campaign for a UN World Citizens' Initiative
The UN's democratic deficit
The United Nations is an exclusive club of representatives of UN Member States. Its democratic deficit has been complained about for decades. While the UN has made some effort to include representatives of civil society and other major groups in some of its deliberations, there are no means for ordinary citizens to take influence.
Examples for citizens' input
In many countries there are instruments that allow citizens to provide input for consideration of the executive or legislative branch of government. In the European Union there is the official instrument of a European Citizens' Initiative (ECI) which gives citizens the power to submit a legislative proposal to the European Commission.
A World Citizens' Initiative
The idea of a UN World Citizens' Initiative (UNWCI) is that if a certain number of global citizens endorses a citizen-launched initiative, UN bodies such as the General Assembly or the Security Council have to put the item on their agenda and give representatives of the initiative the floor to make their case. A UNWCI will allow global citizens to have their voices heard.
Join the global campaign
Democracy Without Borders in collaboration with Democracy International and CIVICUS: World Alliance for Citizen Participation as well as other civil society organizations across the world are pushing forward a global campaign that advocates the creation of a UN World Citizens' Initiative. We invite you to join our efforts.
www.worldcitizensinitiative.org
Democracy | Opinion
Renewing democracy: proposals and ideas from civil society
Mandeep Tiwana 09.11.2018
UNWCI
World Citizens' Initiative promoted at international civil society week
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All our work is carried out using top of the range car body repair equipment and latest IT platforms. The gallery below shows some of the facilities at CMBB.
Below are a range of vehicles repaired by Cheadle Motor Body Builders.
Specialists in respraying and colour coding of any make of vehicle. Giving that personal touch to your vehicle.


text:
Nanotechnology research leads to car powered by its own body panels
(Nanowerk News) A car powered by its own body panels could soon be driving on our roads after a breakthrough in nanotechnology research by a Queensland University of Technology (QUT) team.
Researchers have developed lightweight "supercapacitors" that can be combined with regular batteries to dramatically boost the power of an electric car.
The discovery was made by Postdoctoral Research Fellow Dr Jinzhang Liu, Professor Nunzio Motta and PhD researcher Marco Notarianni, from QUT's Science and Engineering Faculty - Institute for Future Environments, and PhD researcher Francesca Mirri and Professor Matteo Pasquali, from Rice University in Houston, in the United States.
QUT's Professor Nunzio Motta with one of the university's powerful nanotechnology microscopes.
The supercapacitors - a "sandwich" of electrolyte between two all-carbon electrodes - were made into a thin and extremely strong film with a high power density.
The film could be embedded in a car's body panels, roof, doors, bonnet and floor - storing enough energy to turbocharge an electric car's battery in just a few minutes.
The findings, published in the Journal of Power Sources ("High performance all-carbon thin film supercapacitors") and the Nanotechnology journal ("Graphene-based supercapacitor with carbon nanotube film as highly efficient current collector"), mean a car partly powered by its own body panels could be a reality within five years, Mr Notarianni said.
"Vehicles need an extra energy spurt for acceleration, and this is where supercapacitors come in. They hold a limited amount of charge, but they are able to deliver it very quickly, making them the perfect complement to mass-storage batteries," he said.
"Supercapacitors offer a high power output in a short time, meaning a faster acceleration rate of the car and a charging time of just a few minutes, compared to several hours for a standard electric car battery."
Dr Liu said currently the "energy density" of a supercapacitor is lower than a standard lithium ion (Li-Ion) battery, but its "high power density", or ability to release power in a short time, is "far beyond" a conventional battery.
"Supercapacitors are presently combined with standard Li-Ion batteries to power electric cars, with a substantial weight reduction and increase in performance," he said.
"In the future, it is hoped the supercapacitor will be developed to store more energy than a Li-Ion battery while retaining the ability to release its energy up to 10 times faster - meaning the car could be entirely powered by the supercapacitors in its body panels.
"After one full charge this car should be able to run up to 500km - similar to a petrol-powered car and more than double the current limit of an electric car."
Dr Liu said the technology would also potentially be used for rapid charges of other battery-powered devices.
"For example, by putting the film on the back of a smart phone to charge it extremely quickly," he said.
The discovery may be a game-changer for the automotive industry, with significant impacts on financial, as well as environmental, factors.
"We are using cheap carbon materials to make supercapacitors and the price of industry scale production will be low," Professor Motta said.
"The price of Li-Ion batteries cannot decrease a lot because the price of Lithium remains high. This technique does not rely on metals and other toxic materials either, so it is environmentally friendly if it needs to be disposed of."
Source: Queensland University of Technology


text:
As National Summer Learning Week concludes, local programs reflect on the value of celebrating summer learning | Local Education
Subscribe today and support local journalism!
Across the Sky podcast: Meet the meteorologists from the Lee Weather Team!
Photos: Stepping Up for Summer Learning event at the TaxSlayer Center
Many celebrate summer as a break from school and learning.
But this past week, kids and educators nationwide celebrated National Summer Learning Week.
Locally, over 800 summer camp children attended the "Stepping Up for Summer Learning" event at the TaxSlayer Center in Moline — the largest summer learning celebration in Iowa and Illinois.
Indy Learning Team, MLK Center program targets low literacy rates - WISH-TV | Indianapolis News | Indiana Weather
The celebration, which ran from July 11 to 15, was launched by the National Summer Learning Association (NSLA) — a nationwide nonprofit investing in summer learning to help close the achievement gap.
Spring Forward, a local after-school and summer camp program, partnered with the NSLA to host Monday's event. Dan McNeil, the program's director, wants people to understand that there's an industry of summer learning — one that involves school districts, nonprofits and others who hope to keep kids engaged during the summer.
Over 800 QC summer camp children attended the "Stepping Up for Summer Learning" event to kick off National Summer Learning Week on Monday, July 11. This was the largest summer learning celebration in Iowa and Illinois.
"We work with so many partners who offer so much," he said. "We had close to 60 teachers that worked our camps; we're focused on summer learning, so those certified teachers are important. A good summer program combines academics with camp activities, games, play, and we try to do that to the best of our ability."
The Rock Island Public Library runs a summer reading challenge from the beginning of June to the end of July, where participants can earn activity badges and prizes after completing reading goals.
Lisa Lockheart, outreach liaison for the library, said nationwide celebrations like National Summer Learning Week emphasized the importance of continuing to learn and read during the summer months.
"I think the national attention really does reinforce the importance that, while summer is for fun, learning can also be fun," she said. "Leaning is something that needs to continue … and there are all different types of learning going on in the events we offer."
Lockheart said the goal of the summer reading challenge was to have all participants read 100,000 minutes. Last she checked, participants had read 235,780 minutes.
Sheri Peacock is the associate superintendent of student achievement for the East Moline School District. Her role coordinates all internal or collaborative summer programming for the district.
Peacock views summer learning as a worthwhile and rewarding investment.
"Anyone in the community, whether you're a taxpayer or business owner, can invest in summer learning. Either donating, volunteering or just being aware it's happening in your community because these are your students," she said. "If you invest in kids now, they'll be the ones who will take care of the community. It will pay off not only now, but in the future."
Lockheart said the collaboration between summer learning programs and organizations shone a spotlight on how valuable of an investment summer learning was.
"It really makes a statement about the resources that we have available in our community, and we love participating in it," she said. "We all bring something to the table and the more we work together, the better off we are."
Partnerships between summer programs and school districts are beneficial, too, said Two Rivers YMCA Camp Director Cami Ill.
"A lot of that loss happens over the summer when kids don't have access to books or things they can use to keep up. We partner with districts so we can identify kids who might need some extra help," she said.
Ill said she was happy to see so many new faces this summer as the world of education still recovered from the pandemic.
"This summer felt a little more natural. They've had a year of going back to school," she said. "I was happy we were able to get back to normal and take more kids — because the need was there."
McNeil and Peacock agree that summer learning programs expand access to academic achievement — and fun — for all student demographics.
"Summer is sometimes considered the most unequal time in America because a lot of our kids don't have the same experiences as their more affluent peers. Some families are going on vacations or have sports, but some kids aren't," McNeil said. "Our camps are about equity, making sure every child has an amazing summer experience."
Sometimes, summer learning programs provide kids with experiences that expand their worldview.
"Let's say someone came over from the Putnam to do a program. It might have touched upon something one of the children sitting in that room is passionate about — they might be the next museum curator," Peacock said. "You can open the world up for a child through these experiences."
McNeil said Spring Forward was wrapping up its summer program with a pool party Sunday at Whitewater Junction in Rock Island. Campers and their families who attended at least 80% of the camp will be eligible to participate — because just like school, summer learning attendance is important.
Journey Green, 9, leafs through a book during the Stepping Up for Summer Learning event Monday, July 11, 2022, at the TaxSlayer Center in Moline.
NIKOS FRAZIER
Nate DeMarlie, 12, jumps on an airbag to launch a rocket during the Stepping Up for Summer Learning event, Monday, July 11, 2022, at the TaxSlayer Center in Moline.
Scenes from the Stepping Up for Summer Learning event, Monday, July 11, 2022, at the TaxSlayer Center in Moline.
Steamwheelers' Jaylin Swan runs a drill with campers during the Stepping Up for Summer Learning event, Monday, July 11, 2022, at the TaxSlayer Center in Moline.
Stepping Up for Summer Learning event was held at the TaxSlayer Center in Moline on Monday and kicked off National Summer Learning Week.
Dan McNeil, executive director of Spring Forward, speaks during the Stepping Up for Summer Learning event, Monday, July 11, 2022, at the TaxSlayer Center in Moline.
Alannah Herrera, 10, reaches out to choose a book to take home during the Stepping Up for Summer Learning event, Monday, July 11, 2022, at the TaxSlayer Center in Moline.
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Within Teamwork Projects, any file that has been attached to a task can also be removed at a later date.
To detach a file from a task, navigate to the Tasks area of your project.
Locate the task which has the file attached and select it. You will then see an option to Manage Task Attachments.
When you select this, you will see a checkbox option next to each file that have been attached. To remove a file, uncheck the box next to the file and select Save Changes.


It seems like it was not long ago that Kent and I presented Preparing for the Future of SCCM and Maximizing SCCM 1511 for Enterprise Client. But that was a year ago and so much has happened since then. We thought it was worth taking another look at where System Center Configuration Management is and where it's heading which we did in this recent webinar.
A lot has changed since then. We have seen three production releases of Current Branch (1602, 1606, and 1610) and the 1511 version that we talked about last time is no longer in support. The Configuration Manager Team was also busy releasing monthly Technical Previews, as well as a new Microsoft Deployment Toolkit release (build 8443). This is a far cry from the "old days" of System Center Configuration Management updates every few years!
How to get updates has changed. So has how to upgrade to the next version.
We now have the concept of Baseline Builds and In-console Updates. Baseline Builds are essentially full installs that you use to get to Current Branch from Configuration Manager 2012 or if installing a brand new Current Branch site. In-console Updates are released in between the Baseline Builds and show up directly in the Configuration Manager Console. Either type can be a "Fast Ring" or "Slow Ring" release. This is when the new build shows up in the console.
We also talked about and showed some of the key features in each of the Current Branch releases.
Along with a new release cycle comes a new support cycle. Current Branch builds are supported for exactly one year from release. This means that companies are going to need to upgrade at least once per year in order to stay supported. However, the goal is to get companies to upgrade with every release and make the process as quick and easy as possible. Moving from version to version is easier than applying a service pack. And a testament to back this up, as of December 2016, over ten thousand unique customers were already running version 1610.
If you are still using System Center 2012 Configuration Manager, (hopefully, SP2 or R2 SP1), mainstream support ended in July of 2016. Keep in mind that it only supports deploying the first two releases of Windows 10. One of those versions, Windows 10 1507, is no longer supported after March of this year.
For more information on the support, life-cycle see: Support for System Center Configuration Manager current branch versions.
These have addressed issues that were identified during the Fast Ring period, but there is another, somewhat major issue that still exists. This one happens to be with the Apply Driver Package step failing (in some cases) when the ADK has been upgraded to ADK 1607 on Configuration Manager Current Branch 1602 or newer. The Configuration Manager OSD Support Team's Frank Rojas has published an excellent blog on the issue (Apply Driver Package task fails when the ADK is upgraded to ADK 10 1607) and he provides three work around solutions until the problem is fixed. This will likely come with an update to the ADK since the problem appears to be with DISM.
Kent has a neat method for handling drivers that he demonstrated. Microsoft MVP Kim Oppalfens expanded on this method and made it dynamic in his blog post.
If you would like to see something more about CM, head on over to the System Center Configuration Manager Feedback site. This is where you can vote on feature ideas that have already been submitted. Or, you can submit new ideas that you would like to see in a future version of the product. The voting feedback enables to the Configuration Manager Team to prioritize work on feature ideas.
As for 2017, we are likely to see two to three more Configuration Manager Current Branch releases. Possibly two Windows 10 releases! We are especially looking forward to the highly-anticipated Windows 10 Creators Update release. Hopefully, you enjoyed the webinar. Be sure to check out the follow-up Questions and Answers blog.