Toward Repurposing Ciclopirox as an Antibiotic against Drug-Resistant Acinetobacter baumannii,Escherichia coli,and Klebsiella pneumoniae

Antibiotic-resistant infections caused by gram-negative bacteria are a major healthcare concern. Repurposing drugs circumvents the time and money limitations associated with developing new antimicrobial agents needed to combat these antibiotic-resistant infections. Here we identified the off-patent antifungal agent, ciclopirox, as a candidate to repurpose for antibiotic use. To test the efficacy of ciclopirox against antibiotic-resistant pathogens, we used a curated collection of Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae clinical isolates that are representative of known antibiotic resistance phenotypes. We found that ciclopirox, at 5–15 µg/ml concentrations, inhibited bacterial growth regardless of the antibiotic resistance status. At these same concentrations, ciclopirox reduced growth of Pseudomonas aeruginosa clinical isolates, but some of these pathogens required higher ciclopirox concentrations to completely block growth. To determine how ciclopirox inhibits bacterial growth, we performed an overexpression screen in E. coli. This screen revealed that galE, which encodes UDP-glucose 4-epimerase, rescued bacterial growth at otherwise restrictive ciclopirox concentrations. We found that ciclopirox does not inhibit epimerization of UDP-galactose by purified E. coli GalE; however, ΔgalU, ΔgalE, ΔrfaI, or ΔrfaB mutant strains all have lower ciclopirox minimum inhibitory concentrations than the parent strain. The galU, galE, rfaI, and rfaB genes all encode enzymes that use UDP-galactose or UDP-glucose for galactose metabolism and lipopolysaccharide (LPS) biosynthesis. Indeed, we found that ciclopirox altered LPS composition of an E. coli clinical isolate. Taken together, our data demonstrate that ciclopirox affects galactose metabolism and LPS biosynthesis, two pathways important for bacterial growth and virulence. The lack of any reported fungal resistance to ciclopirox in over twenty years of use in the clinic, its excellent safety profiles, novel target(s), and efficacy, make ciclopirox a promising potential antimicrobial agent to use against multidrug-resistant problematic gram-negative pathogens.     

Selection of Reference Genes for Real-Time Quantitative PCR in Pinus massoniana Post Nematode Inoculation

Pinus massoniaia Lamb has gained more and more attention as the most important tree species for timber and forestation in South China. Gene expression studies are of great importance to identify new and elite cultivars. Real-time quantitative PCR, a highly sensitive and specific method, is commonly used in the analysis of gene expression. The appropriate reference genes must be employed to normalize the calculation program for ascertaining repeatable and significant results. Herein, eleven housekeeping genes were evaluated during different stages of P. massoniana post nematode inoculation in this study. Three statistical approaches such as geNorm, NormFinder and BestKeeper were selected to analyze the stability of candidate genes. The results indicated that U2af and β-TUB were the most stable reference genes. These two genes could be used for the normalization in most of the experiments of P. massoniana, while Histone and AK were the least stable ones. In addition, EF expressed at the lowest average Ct value was the most abundant candidate gene. As an important gene associated with defense mechanisms, ABC transporter was analyzed by qRT-PCR, and the results were used to confirm the reliability of two genes. The selected reference genes in the present study will be conducive to future gene expression normalized by qRT-PCR in P. massoniana.     

The primate extinction crisis in China: immediate challenges and a way forward

China is facing an unprecedented set of challenges in balancing the effects of economic development and global climate change with environmental protection and maintaining biodiversity. Although positive steps have been undertaken to remedy this situation, currently 80% of China’s 25 extant primate species are threatened, 15–18 species have population sizes of less than 3000 individuals, and two species of gibbons and one species of langur have been extirpated over the past few decades. Today, virtually all species of primates in China inhabit fragmented landscapes and are distributed in small isolated subpopulations with limited opportunities to exchange individuals or genetic information. Here we present a historical framework examining how human-induced environmental changes, particularly since the second half of the 20th century, accelerated primate population decline in China. In addition, we modeled the expected spatial conflict between agricultural expansion and primate distributions over the next 25–75 years and assessed the current overlap between protected areas and primate distributions. Depending on the assumptions of the spatial conflict model, primate distributions are expected to decline by an additional 51–87% by the year 2100. Thus, unless large-scale conservation policies are implemented immediately the current trend of primate population decline, local extirpation, and species extinctions will accelerate. To mitigate against such extinction scenarios, we advocate the creation of a Chinese national agency and repository of environmental information focused on public awareness and education, the implementation of targeted programs of habitat restoration designed to return impacted forests to a more natural state especially within and at the boundaries of nature reserves, the establishment of additional protect areas, and the construction of a latticework of corridors connecting isolated primate subpopulations. This comprehensive approach offers the most effective way to protect China’s animal and plant biodiversity, including its endangered primate populations.     

High-amylose rice improves indices of animal health in normal and diabetic rats

A high-amylose rice with 64.8% amylose content (AC) was developed by transgenic inhibition of two isoforms of starch branching enzyme (SBE), SBEI and SBEIIb, in an indica rice cultivar. The expression of SBEI and SBEIIb was completely inhibited in the transgenic line, whereas the expression of granule-bound starch synthase was normal. Compared with wild-type rice, drastic reductions in both SBEs in the transgenic rice increased apparent AC in flour from 27.2% to 64.8%, resistant starch (RS) content from 0% to 14.6% and total dietary fibre (TDF) from 6.8% to 15.2%. Elevated AC increased the proportion of long unit chains in amylopectin and increased onset gelatinization temperature and resistance to alkaline digestion; however, kernel weight was decreased. A rat feeding trial indicated that consumption of high-amylose rice decreased body weight gain significantly (P < 0.01); increased faecal mass, faecal moisture and short-chain fatty acids; and lowered the faecal pH. An acute oral rice tolerance test revealed that the high-amylose rice had a positive effect on lowering the blood glucose response in diabetic Zucker fatty rats. This novel rice with its high AC, RS and TDF offers potential benefits for its use in foods and in industrial applications.     

Crystal structure of the E2 domain of amyloid precursor protein-like protein 1 in complex with sucrose octasulfate

Missense mutations in the amyloid precursor protein (APP) gene can cause familial Alzheimer disease. It is thought that APP and APP-like proteins (APLPs) may play a role in adhesion and signal transduction because their ectodomains interact with components of the extracellular matrix. Heparin binding induces dimerization of APP and APLPs. To help explain how these proteins interact with heparin, we have determined the crystal structure of the E2 domain of APLP1 in complex with sucrose octasulfate (SOS). A total of three SOS molecules are bound to the E2 dimer. Two SOSs are bound inside a narrow intersubdomain groove, and the third SOS is bound near the two-fold axis of the protein. Mutational analyses show that most residues interacting with SOS also contribute to heparin binding, although in varying degrees; a deep pocket, defined by His-376, Lys-422, and Arg-429, and an interfacial site between Lys-314 and its symmetry mate are most important in the binding of the negatively charged polysaccharide. Comparison with a lower resolution APP structure shows that all key heparin binding residues are conserved and identically positioned, suggesting that APLP1 and APP may bind heparin similarly. In transfected HEK-293 cells, mutating residues responsible for heparin binding causes little change in the proteolysis of APP by the secretases. However, mutating a pair of conserved basic residues (equivalent to Arg-414 and Arg-415 of APLP1) immediately adjacent to the heparin binding site affects both the maturation and the processing of APP.     

Glucagon gene polymorphism modifies the effects of smoking and physical activity on risk of type 2 diabetes mellitus in Han Chinese

Few genome-wide association studies have considered interactions between multiple genetic variants and environmental factors associated with disease. The interaction was examined between a glucagon gene (GCG) polymorphism and smoking, alcohol consumption and physical activity and the association with risk of type 2 diabetes mellitus (T2DM) in a case–control study of Chinese Han subjects. The rs12104705 polymorphism of GCG and interactions with environmental variables were analyzed for 9619 participants by binary multiple logistic regression. Smoking with the C-C haplotype of rs12104705 was associated with increased risk of T2DM (OR = 1.174, 95% CI = 1.013–1.361). Moderate and high physical activity with the C-C genotype was associated with decreased risk of T2DM as compared with low physical activity with the genotype (OR = 0.251, 95% CI = 0.206–0.306 and OR = 0.190, 95% CI = 0.164–0.220). However, the interaction of drinking and genotype was not associated with risk of T2DM. Genetic polymorphism in rs12104705 of GCG may interact with smoking and physical activity to modify the risk of T2DM.     

Failure of PCR to Detect Treponema pallidum ssp. pertenue DNA in Blood in Latent Yaws

Yaws, caused by Treponema pallidum ssp. pertenue, is a neglected tropical disease closely related to venereal syphilis and is targeted for eradication by 2020. Latent yaws represents a diagnostic challenge, and current tools cannot adequately distinguish between individuals with true latent infection and individuals who are serofast following successful treatment. PCR on blood has previously been shown to detect T. pallidum DNA in patients with syphilis, suggesting that this approach may be of value in yaws. We performed real-time PCR for Treponema pallidum ssp. pertenue on blood samples from 140 children with positive T. pallidum Particle Agglutination (TPPA) and Rapid Plasma Reagin (RPR) tests and 7 controls (negative serology), all collected as part of a prospective study of yaws in the Solomon Islands. All samples were also tested by a nested PCR for T. pallidum. 12 patients had clinical evidence of active yaws whilst 128 were considered to have latent yaws. 43 children had high titre rapid plasma reagins (RPRs) of ≥1:32. PCR testing with both assays gave negative results in all cases. It is possible that the failure to detect T. pallidum ssp. pertenue in blood reflects lower loads of organism in latent yaws compared to those in latent infection with T. pallidum ssp. pertenue, and/or a lower propensity for haematogenous dissemination in yaws than in syphilis. As the goal of the yaws control programme is eradication, a tool that can differentiate true latent infection from individuals who are serofast would be of value; however, PCR of blood is not that tool.     

Design,synthesis, and biological evaluation of novel water-soluble triptolide derivatives: Antineoplastic activity against imatinib-resistant CML cells bearing T315I mutant Bcr-Abl

Imatinib (STI571) is the frontline targeted-therapeutic agent for patients with chronic myelogenous leukemia (CML). However, resistance to imatinib due to point mutations in Bcr-Abl kinase domain is an emerging problem. We recently reported that triptolide (compound 1) could effectively kill CML cells including those harboring T315I mutant Bcr-Abl. In the present study, we designed a series of C-14 triptolide derivatives with C-14-hydroxyl substituted by different amine esters (3–18): 3–6 and 13 (by aliphatic chain amine esters); 7–9, 11, 12 and 15–18 (by alicyclic amine esters with different size), and 10 and 14 (by aralkylamine esters).The compounds were examined for their antineoplastic activity against CML cells (including KBM5-T315I cells) in terms of proliferation inhibition, apoptosis and signal transduction. Nude mouse xenograft model was also used to evaluate the in vivo activity. Compounds 2–9, 11–14, 17 and 18 exhibited a potent inhibitory activity against KBM5 and KBM5-T315I cells. This series of derivatives down-regulated Bcr-Abl mRNA. Compounds 4, 5, 8 and 9 were further examined for their impact on signaling and apoptosis with immunoblotting. Compound 5 was chosen for evaluation in a nude mouse xenograft model. The stereo-hindrance of C-14 group appeared to be responsible for the antitumor effect. The computational small molecule-protein docking analysis illustrated the possible interaction between compound 9 and RNA polymerase II. Our results suggest that this series of derivatives may be promising agents to overcome imatinib-resistance caused by the Bcr-Abl-T315I mutation.