Chemical Synergy between Ionophore PBT2 and Zinc Reverses Antibiotic Resistance.

The World Health Organization reports that antibiotic-resistant pathogens represent an imminent global health disaster for the 21st century. Gram-positive superbugs threaten to breach last-line antibiotic treatment, and the pharmaceutical industry antibiotic development pipeline is waning. Here we report the synergy between ionophore-induced physiological stress in Gram-positive bacteria and antibiotic treatment. PBT2 is a safe-for-human-use zinc ionophore that has progressed to phase 2 clinical trials for Alzheimer's and Huntington's disease treatment. In combination with zinc, PBT2 exhibits antibacterial activity and disrupts cellular homeostasis in erythromycin-resistant group A Streptococcus (GAS), methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus (VRE). We were unable to select for mutants resistant to PBT2-zinc treatment. While ineffective alone against resistant bacteria, several clinically relevant antibiotics act synergistically with PBT2-zinc to enhance killing of these Gram-positive pathogens. These data represent a new paradigm whereby disruption of bacterial metal homeostasis reverses antibiotic-resistant phenotypes in a number of priority human bacterial pathogens.IMPORTANCE The rise of bacterial antibiotic resistance coupled with a reduction in new antibiotic development has placed significant burdens on global health care. Resistant bacterial pathogens such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus are leading causes of community- and hospital-acquired infection and present a significant clinical challenge. These pathogens have acquired resistance to broad classes of antimicrobials. Furthermore, Streptococcus pyogenes, a significant disease agent among Indigenous Australians, has now acquired resistance to several antibiotic classes. With a rise in antibiotic resistance and reduction in new antibiotic discovery, it is imperative to investigate alternative therapeutic regimens that complement the use of current antibiotic treatment strategies. As stated by the WHO Director-General, "On current trends, common diseases may become untreatable. Doctors facing patients will have to say, Sorry, there is nothing I can do for you.

Overexpression of defense response genes in transgenic wheat enhances resistance to Fusarium head blight

Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum and other Fusarium species, is a major disease problem for wheat production worldwide. To combat this problem, large-scale breeding efforts have been established. Although progress has been made through standard breeding approaches, the level of resistance attained is insufficient to withstand epidemic conditions. Genetic engineering provides an alternative approach to enhance the level of resistance. Many defense response genes are induced in wheat during F. graminearum infection and may play a role in reducing FHB. The objectives of this study were (1) to develop transgenic wheat overexpressing the defense response genes α-1-purothionin, thaumatin-like protein 1 (tlp-1), and β-1,3-glucanase; and (2) to test the resultant transgenic wheat lines against F. graminearum infection under greenhouse and field conditions. Using the wheat cultivar Bobwhite, we developed one, two, and four lines carrying the α-1-purothionin, tlp-1, and β-1,3-glucanase transgenes, respectively, that had statistically significant reductions in FHB severity in greenhouse evaluations. We tested these seven transgenic lines under field conditions for percent FHB disease severity, deoxynivalenol (DON) mycotoxin accumulation, and percent visually scabby kernels (VSK). Six of the seven lines differed from the nontransgenic parental Bobwhite line for at least one of the disease traits. A β-1,3-glucanase transgenic line had enhanced resistance, showing lower FHB severity, DON concentration, and percent VSK compared to Bobwhite. Taken together, the results showed that overexpression of defense response genes in wheat could enhance the FHB resistance in both greenhouse and field conditions

Biochemical correlation of activity of the α-dystroglycan-modifying glycosyltransferase POMGnT1 with mutations in muscle-eye-brain disease

Congenital muscular dystrophies have a broad spectrum of genotypes and phenotypes and there is a need for a better biochemical understanding of this group of diseases in order to aid diagnosis and treatment. Several mutations resulting in these diseases cause reduced O-mannosyl glycosylation of glycoproteins, including α-dystroglycan. The enzyme POMGnT1 (protein-O-mannose N-acetylglucosaminyltransferase 1; EC 2.4.1.-) catalyses the transfer of N-acetylglucosamine to O-linked mannose of α-dystroglycan. In the present paper we describe the biochemical characterization of 14 clinical mutants of the glycosyltransferase POMGnT1, which have been linked to muscle-eye-brain disease or similar conditions. Truncated mutant variants of the human enzyme (recombinant POMGnT1) were expressed in Escherichia coli and screened for catalytic activity. We find that three mutants show some activity towards mannosylated peptide substrates mimicking α-dystroglycan; the residues affected by these mutants are predicted by homology modelling to be on the periphery of the POMGnT1 surface. Only in part does the location of a previously described mutated residue on the periphery of the protein structure correlate with a less severe disease mutant

Antiangiogenic platinum through glycan targeting

Heparan sulfate is identified as a ligand receptor for polynuclear platinum anti-cancer agents through sulfate cluster binding. We present a new biological role for platinum and coordination compounds and a new target for metal-based drugs while presenting a new chemotype for heparanase and growth factor inhibition through modulation (metalloshielding) of their interactions. Masking of extracellular (ECM)-resident heparan sulfate (HS) through metalloshielding results in very effective inhibition of physiologically critical HS functions including enzyme (heparanase, HPSE) and protein growth factor recognition. The interaction of the highly cationic polynuclear platinum complexes (PPCs) with the highly sulfated pentasaccharide Fondaparinux (FPX, in this case as a model HS-like substrate) results in inhibition of its cleavage by the HS-related enzyme heparanase. Binding of the fibroblast growth factor FGF-2 to HS is also inhibited with consequences for downstream signalling events as measured by a reduction in accumulation of phospho-S6 ribosomal protein in human colon tumor HCT-116 cells. The end-point of inhibition of HPSE activity and growth factor growth factor signaling is the prevention of cell invasion and angiogenesis. Finally these events culminate in inhibition of HCT-116 cell invasion at sub-cytotoxic concentrations and the process of angiogenesis. A competition assay shows that Fondaparinux can sequester the 8+ TriplatinNC from bound DNA, emphasising the strength of PPC–HS interactions. Altering the profile of platinum agents from cytotoxic to anti-metastatic has profound implications for future directions in the development of platinum-based chemotherapeutics.This work was supported by grants from NIH (RO1CA78754) to NPF and The Australian Research Council (DP150100308) to SBP, MvI and NPF and from the Australian National Health and Medical Research Council (455395) to CRP. Support from The Massey Cancer Center CA016059 is gratefully acknowledged

Comparative Analysis of Two Gene-Targeting Approaches Challenges the Tumor-Suppressive Role of the Protein Kinase MK5/PRAK

<div><p>MK5 (MAPK-activated protein kinase 5) or PRAK (p38-regulated and -activated kinase) are alternative names for a serine/threonine protein kinase downstream to ERK3/4 and p38 MAPK. A previous gene targeting approach for MK5/PRAK (termed here MK5/PRAK-Δex8) revealed a seemingly tumor-suppressive role of MK5/PRAK in DMBA-induced one step skin carcinogenesis and Ras-induced transformation. Here we demonstrate that an alternative targeting strategy of MK5/PRAK (termed MK5/PRAK-Δex6) increased neither tumor incidence in the one step skin carcinogenesis model, nor Ras-induced transformation in primary cells. Interestingly, due to the targeting strategies and exon skipping both knockouts do not completely abolish the generation of MK5/PRAK protein, but express MK5/PRAK deletion mutants with different biochemical properties depending on the exon targeted: Targeting of exon 6 leads to expression of an unstable cytoplasmic catalytically inactive MK5/PRAK-Δex6 mutant while targeting of exon 8 results in a more stable nuclear MK5/PRAK-Δex8 mutant with residual catalytic activity. The different properties of the MK5/PRAK deletion mutants could be responsible for the observed discrepancy between the knockout strains and challenge the role of MK5/PRAK in p53-dependent tumor suppression. Further MK5/PRAK knockout and knock-in mouse strains will be necessary to assign a physiological function to MK5/PRAK in this model organism.</p></div

Proficiency testing of PIK3CA mutations in HR+/HER2-breast cancer on liquid biopsy and tissue

Precision oncology based on specific molecular alterations requires precise and reliable detection of therapeutic targets in order to initiate the optimal treatment. In many European countries-including Germany-assays employed for this purpose are highly diverse and not prescribed by authorities, making inter-laboratory comparison difficult. To ensure reproducible molecular diagnostic results across many laboratories and different assays, ring trials are essential and a well-established tool. Here, we describe the design and results of the ring trial for the detection of therapeutically relevant PIK3CA hotspot mutations in HR+/HER2-breast cancer tissue and liquid biopsy (LB). For PIK3CA mutation detection in tissue samples, 43 of the 54 participants (80%) provided results compliant with the reference values. Participants using NGS-based assays showed higher success rate (82%) than those employing Sanger sequencing (57%). LB testing was performed with two reference materials differing in the length of the mutated DNA fragments. Most participants used NGS-based or commercial realtime PCR assays (70%). The 167 bp fragments led to a successful PIK3CA mutation detection by only 31% of participants whereas longer fragments of 490 bp were detectable even by non-optimal assays (83%). In conclusion, the first ring trial for PIK3CA mutation detection in Germany showed that PIK3CA mutation analysis is broadly established for tissue samples and that NGS-based tests seem to be more suitable than Sanger sequencing. PIK3CA mutation detection in LB should be carried out with assays specifically designed for this purpose in order to avoid false-negative results

Detection of protein mutants of MK5/PRAK and analysis of their stability.

<p>A) Detection of MK5/PRAK proteins in lysates from primary Δex6 and Δex8 MEFs. Specificity of the signal is demonstrated by MK5/PRAK siRNA treatment. Loading control was performed by Western blot of elongation factor 2 (EF2). According to the molecular weight marker shown left, the three MK5/PRAK specific bands, which are reduced by MK5/PRAK siRNA treatment, migrate with apparent molecular masses of about 66 kDa, 52 kDa and 50 kDa (double band). The 52 kDa band fits best to the calculated mass of full length MK5/PRAK (1–473) of 54,152 Da. The 50 kDa band could represent the 471 amino acid splice variant lacking “GK” [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136138#pone.0136138.ref030" target="_blank">30</a>], since exchange or loss of one or a few charged amino acids (“K”) often changes migration in SDS-PAGE for a few kDa. As also described for endogenous mouse MK2/MAPKAPK2, where an additional band of about 56 kDa is detected in addition to the band corresponding to the calculated mass of 44,050 Da for mouse MK2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136138#pone.0136138.ref038" target="_blank">38</a>], there is a band detected for endogenous (but not overexpressed) mouse MK5/PRAK which migrates significantly slower than expected from the calculated molecular mass (66 kDa instead 52 KDa). As for MK2, we do not know the reason for this slower migrating band of MK5/PRAK so far. B, C) Stability of endogenous MK5/PRAK proteins was monitored by Western blot (B) and chemiluminescence quantification (C) at different times after cycloheximide (CHX) treatment of MEFs. The program ImageJ 1.38x (NIH- <a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a>) was used for chemiluminescence quantification.</p

Kinase activity of wild type MK5/PRAK, MK5/PRAK-Δex6 and -Δex8.

<p>A) HEK293 cells were transfected with the constructs indicated. The total amount of DNA per well was kept constant (1,6μg DNA per well in 12-well plate) by adding pcDNA-flag expression vector. 24 h post transfection cells were lysed in the plates with 1x Laemmli buffer. Lysates were analyzed by Western blotting using the indicated antibodies. A significant increase in phosphorylation of the substrate Hsp27 was only detected for WT MK5/PRAK in the presence of overexpressed p38 MAPK, ERK3 or ERK4. B) HEK293 cells were transfected with the expression constructs indicated. 24 h post transfection the cells were lysed and GFP-tagged MK5/PRAK or its exon 6 (Δex6) or exon 8 (Δex8) deletion mutants were immuno-precipitated by GFP nanobodies coupled to M270 epoxy beads. The beads were used in a kinase reaction with recombinant Hsp27 as a substrate. The reaction mixture was resolved by SDS-PAGE and Hsp27 phosphorylating activity was detected by phospho-imaging (autoradiogram) and Western blot against pS82-Hsp27. Increased Hsp27-kinase activity is only detected for WT MK5/PRAK (asterisk). C) In vitro MK5/PRAK kinase assay with Hsp27 as substrates. GST–p38 was used to activate MK5/PRAK in vitro. While Hsp27-kinase activity is only detected for WT MK5/PRAK, auto-phosphorylating activity in the absence of p38 MAPK (left panel) is detected for both WT MK5/PRAK and Δex8 (asterisk).</p

Altered subcellular localization of GFP- MK5-Δex6, but not of GFP- MK5-Δex8, in HEK293 cells and effect of co-expression of ERK3.

<p>GFP and GFP-fusions of MK5/PRAK and its mutants were expressed in HEK293 cells in the absence or presence of co-expressed red fluorescent protein (RFP)-fused ERK3 and subcellular localization was followed by confocal fluorescence microscopy.</p

Detection of mutant MK5/PRAK mRNAs in Δex6 and Δex8 MEFs.

<p>A) MK5/PRAK gene structure and assignment of the conserved protein kinase subdomains, the overlapping nuclear export (NES)/ nuclear localization signal (NLS) and the ERK3/4-binding site [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136138#pone.0136138.ref006" target="_blank">6</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136138#pone.0136138.ref037" target="_blank">37</a>] to the different exons. B) Detection of MK5/PRAK mRNA fragments from Δex6 and Δex8 MEFs by exon specific PCR.</p