In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms. Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils. This process has the potential to be destructive to tissues if excessive or held in an unresolved state.  Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration. This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil. Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface. The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1). Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).  The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls. This in vitro model system can be manipulated and applied to other mucosal surfaces. Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections. A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.     

分子模拟在多黏菌素药理机制研究中的应用

多黏菌素是一种膜靶向的脂肽类抗生素，是临床上治疗革兰氏阴性多重耐药菌感染的最后一道防线。通过与脂多糖相互作用，多黏菌素破坏细菌外膜结构并导致细菌死亡。然而，受限于生物化学和结构生物学手段对细胞膜-药物相互作用的表征能力，目前对多黏菌素药理机制的认识还不充分，从而限制了新一代多黏菌素药物的设计和开发。为此，本文总结了近年来利用分子动力学方法对细胞膜系统与多黏菌素相互作用的研究进展，为深入理解多黏菌素药理机制与细胞膜系统的内在联系，加快新型抗生素药物研发提供新思路。     

Scalable Nanohelices for Predictive Studies and Enhanced 3D Visualization

Spring-like materials are ubiquitous in nature and of interest in nanotechnology for energy harvesting, hydrogen storage, and biological sensing applications.  For predictive simulations, it has become increasingly important to be able to model the structure of nanohelices accurately.  To study the effect of local structure on the properties of these complex geometries one must develop realistic models.  To date, software packages are rather limited in creating atomistic helical models.  This work focuses on producing atomistic models of silica glass (SiO₂) nanoribbons and nanosprings for molecular dynamics (MD) simulations. Using an MD model of “bulk” silica glass, two computational procedures to precisely create the shape of nanoribbons and nanosprings are presented.  The first method employs the AWK programming language and open-source software to effectively carve various shapes of silica nanoribbons from the initial bulk model, using desired dimensions and parametric equations to define a helix.  With this method, accurate atomistic silica nanoribbons can be generated for a range of pitch values and dimensions.  The second method involves a more robust code which allows flexibility in modeling nanohelical structures.  This approach utilizes a C++ code particularly written to implement pre-screening methods as well as the mathematical equations for a helix, resulting in greater precision and efficiency when creating nanospring models.  Using these codes, well-defined and scalable nanoribbons and nanosprings suited for atomistic simulations can be effectively created.  An added value in both open-source codes is that they can be adapted to reproduce different helical structures, independent of material.  In addition, a MATLAB graphical user interface (GUI) is used to enhance learning through visualization and interaction for a general user with the atomistic helical structures.  One application of these methods is the recent study of nanohelices via MD simulations for mechanical energy harvesting purposes.     

Transcriptional response of mushrooms to artificial sun exposure

Climate change causes increased tree mortality leading to canopy loss and thus sun‐exposed forest floors. Sun exposure creates extreme temperatures and radiation, with potentially more drastic effects on forest organisms than the current increase in mean temperature. Such conditions might potentially negatively affect the maturation of mushrooms of forest fungi. A failure of reaching maturation would mean no sexual spore release and, thus, entail a loss of genetic diversity. However, we currently have a limited understanding of the quality and quantity of mushroom‐specific molecular responses caused by sun exposure. Thus, to understand the short‐term responses toward enhanced sun exposure, we exposed mushrooms of the wood‐inhabiting forest species Lentinula edodes, while still attached to their mycelium and substrate, to artificial solar light (ca. 30°C and 100,000 lux) for 5, 30, and 60 min. We found significant differentially expressed genes at 30 and 60 min. Eukaryotic Orthologous Groups (KOG) class enrichment pointed to defense mechanisms. The 20 most significant differentially expressed genes showed the expression of heat‐shock proteins, an important family of proteins under heat stress. Although preliminary, our results suggest mushroom‐specific molecular responses to tolerate enhanced sun exposure as expected under climate change. Whether mushroom‐specific molecular responses are able to maintain fungal fitness under opening forest canopies remains to be tested.     

Analysis of diverse β-lactamases presenting high-level resistance in association with OmpK35 and OmpK36 porins in ESBL-producing Klebsiella pneumoniae

Emerging extensively drug-resistant (XDR) Klebsiella pneumoniae due to the production of β-lactamases and porin loss is a substantial worldwide concern. This study aimed to elucidate the role of outer membrane porin (OMP) loss, AmpC, and carbapenemases among extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae strains with XDR phenotype. This study analyzed 79 K. pneumoniae from several clinical sources and detected ESBLs in 29 strains co-harbored with other β-lactamases using standard microbiological practices and phenotypic procedures. Minimum inhibitory concentrations (MICs) were determined against several antibiotics using Microscan WalkAway plus. OMP analysis was carried out using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. ESBL, AmpC, and carbapenemase genes were detected using molecular methods. The microbiological analysis discovered 29 (36.7%) ESBL strains of K. pneumoniae, which showed the co-existence of 7 (24.1%) AmpC β-lactamases and 22 (75.9%) carbapenemases. Porin loss of OmpK35 was observed in 13 (44.8%) and OmpK36 in 8 (27.5%) K. pneumoniae strains. The strains were significantly associated with the intensive care unit (ICU) (p = 0.006) and urinary sources (p = 0.004). The most commonly detected gene variants in each β-lactamase class included 16 (55.2%) bla_CTX-M−1, 7 (100%) bla_CYM-2, 11 (50%) bla_NDM-1, and integron-1 was detected in 21/29 (72.4%) strains. MICs of cephalosporin, fluoroquinolone, carbapenem, aminoglycoside, and β-lactam combinations demonstrated a high number of XDR strains. Tigecycline (2 µg/mL MIC₅₀ and >32 µg/mL MIC₉₀) and colistin (1 µg/mL MIC₅₀ and 8 µg/mL MIC₉₀) presented lower resistance. ESBL K. pneumoniae strains with OmpK35 and OmpK36 porin loss demonstrate conglomerate resistance mechanisms with AmpC and carbapenemases, leading to emerging XDR and pan drug resistance.     

Cryo-EM structure of the fatty acid reductase LuxC–LuxE complex provides insights into bacterial bioluminescence

The discovery of reduced flavin mononucleotide and fatty aldehydes as essential factors of light emission facilitated study of bacterial luminescence. Although the molecular mechanisms underlying bacterial luminescence have been studied for more than 60 years, the structure of the bacterial fatty acid reductase complex remains unclear. Here, we report the cryo-EM structure of the Photobacterium phosphoreum fatty acid reductase complex LuxC–LuxE to a resolution of 2.79 Å. We show that the active site Lys238/Arg355 pair of LuxE is >30 Å from the active site Cys296 of LuxC, implying that catalysis relies on a large conformational change. Furthermore, mutagenesis and biochemical experiments support that the L-shaped cleft inside LuxC plays an important role in substrate binding and reaction. We obtained a series of mutants with significantly improved activity as measured by in vitro bioluminescence assays and demonstrated that the double mutant W111A/F483K displayed the highest activity (370% of the WT). Our results indicated that the activity of LuxC significantly affects the bacterial bioluminescence reaction. Finally, we expressed this mutated lux operon in Escherichia coli but observed that the in vivo concentrations of ATP and NADPH limited the enzyme activity; thus, we conclude that the luminous intensity mainly depends on the level of metabolic energy.     

Allosteric enhancement of the BCR-Abl1 kinase inhibition activity of nilotinib by cobinding of asciminib

Inhibitors that bind competitively to the ATP binding pocket in the kinase domain of the oncogenic fusion protein BCR–Abl1 are used successfully in targeted therapy of chronic myeloid leukemia (CML). Such inhibitors provided the first proof of concept that kinase inhibition can succeed in a clinical setting. However, emergence of drug resistance and dose-dependent toxicities limit the effectiveness of these drugs. Therefore, treatment with a combination of drugs without overlapping resistance mechanisms appears to be an appropriate strategy. In the present work, we explore the effectiveness of combination therapies of the recently developed allosteric inhibitor asciminib with the ATP-competitive inhibitors nilotinib and dasatinib in inhibiting the BCR–Abl1 kinase activity in CML cell lines. Through these experiments, we demonstrate that asciminib significantly enhances the inhibition activity of nilotinib, but not of dasatinib. Exploring molecular mechanisms for such allosteric enhancement via systematic computational investigation incorporating molecular dynamics, metadynamics simulations, and density functional theory calculations, we found two distinct contributions. First, binding of asciminib triggers conformational changes in the inactive state of the protein, thereby making the activation process less favorable by ∼4 kcal/mol. Second, the binding of asciminib decreases the binding free energies of nilotinib by ∼3 and ∼7 kcal/mol for the wildtype and T315I-mutated protein, respectively, suggesting the possibility of reducing nilotinib dosage and lowering risk of developing resistance in the treatment of CML.     

Vegetation resistance and resilience to a decade‐long dry period in the temperate grasslands in China

The duration of climate anomalies has been increasing across the globe, leading to ecosystem function loss. Thus, we need to understand the responses of the ecosystem to long‐term climate anomalies. It remains unclear how ecosystem resistance and resilience respond to long‐term climate anomalies, for example, continuous dry years at a regional scale. Taking the opportunity of a 13‐year dry period in the temperate grasslands in northern China, we quantified the resistance and resilience of the grassland in response to this periodic dry period. We found vegetation resistance to the dry period increased with mean annual precipitation (MAP), while resilience increased at first until at MAP of 250 mm and then decreased slightly. No trade‐off between resistance and resilience was detected when MAP < 250 mm. Our results highlight that xeric ecosystems are most vulnerable to the long‐term dry period. Given expected increases in drought severity and duration in the coming decades, our findings may be helpful to identify vulnerable ecosystems in the world for the purpose of adaptation.     

Construction of a high-density genetic map using specific length amplified fragment markers and identification of a quantitative trait locus for anthracnose resistance in walnut (Juglans regia L.)

Background

Walnut (Juglans regia, 2n = 32, approximately 606 Mb per 1C genome) is an economically important tree crop. Resistance to anthracnose, caused by Colletotrichum gloeosporioides, is a major objective of walnut genetic improvement in China. The recently developed specific length amplified fragment sequencing (SLAF-seq) is an efficient strategy that can obtain large numbers of markers with sufficient sequence information to construct high-density genetic maps and permits detection of quantitative trait loci (QTLs) for molecular breeding.

Results

SLAF-seq generated 161.64 M paired-end reads. 153,820 SLAF markers were obtained, of which 49,174 were polymorphic. 13,635 polymorphic markers were sorted into five segregation types and 2,577 markers of them were used to construct genetic linkage maps: 2,395 of these fell into 16 linkage groups (LGs) for the female map, 448 markers for the male map, and 2,577 markers for the integrated map. Taking into account the size of all LGs, the marker coverage was 2,664.36 cM for the female map, 1,305.58 cM for the male map, and 2,457.82 cM for the integrated map. The average intervals between two adjacent mapped markers were 1.11 cM, 2.91 cM and 0.95 cM for three maps, respectively. ‘SNP_only’ markers accounted for 89.25 % of the markers on the integrated map. Mapping markers contained 5,043 single nucleotide polymorphisms (SNPs) loci, which corresponded to two SNP loci per SLAF marker. According to the integrated map, we used interval mapping (Logarithm of odds, LOD > 3.0) to detect our quantitative trait. One QTL was detected for anthracnose resistance. The interval of this QTL ranged from 165.51 cM to 176.33 cM on LG14, and ten markers in this interval that were above the threshold value were considered to be linked markers to the anthracnose resistance trait. The phenotypic variance explained by each marker ranged from 16.2 to 19.9 %, and their LOD scores varied from 3.22 to 4.04.

Conclusions

High-density genetic maps for walnut containing 16 LGs were constructed using the SLAF-seq method with an F1 population. One QTL for walnut anthracnose resistance was identified based on the map. The results will aid molecular marker-assisted breeding and walnut resistance genes identification.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1822-8) contains supplementary material, which is available to authorized users.     

A ProQ/FinO family protein involved in plasmid copy number control favours fitness of bacteria carrying mcr-1-bearing IncI2 plasmids

The plasmid-encoded colistin resistance gene mcr-1 challenges the use of polymyxins and poses a threat to public health. Although IncI2-type plasmids are the most common vector for spreading the mcr-1 gene, the mechanisms by which these plasmids adapt to host bacteria and maintain resistance genes remain unclear. Herein, we investigated the regulatory mechanism for controlling the fitness cost of an IncI2 plasmid carrying mcr-1. A putative ProQ/FinO family protein encoded by the IncI2 plasmid, designated as PcnR (plasmid copy number repressor), balances the mcr-1 expression and bacteria fitness by repressing the plasmid copy number. It binds to the first stem-loop structure of the repR mRNA to repress RepA expression, which differs from any other previously reported plasmid replication control mechanism. Plasmid invasion experiments revealed that pcnR is essential for the persistence of the mcr-1-bearing IncI2 plasmid in the bacterial populations. Additionally, single-copy mcr-1 gene still exerted a fitness cost to host bacteria, and negatively affected the persistence of the IncI2 plasmid in competitive co-cultures. These findings demonstrate that maintaining mcr-1 plasmid at a single copy is essential for its persistence, and explain the significantly reduced prevalence of mcr-1 following the ban of colistin as a growth promoter in China.     

Effects of Temperature on Resistance in Phaseolus vulgaris Genotypes and on Development of Meloidogyne Species

Phaseolus vulgaris lines with heat-stable resistance to Meloidogyne spp. may be needed to manage root-knot nematodes in tropical regions. Resistance expression before and during the process of nematode penetration and development in resistant genotypes were studied at pre- and postinoculation temperatures of 24 °C and 24 °C, 24 °C and 28 °C, 28 °C and 24 °C, and 28 °C and 28 °C. Resistance was effective at all temperature regimes examined, with fewer nematodes in roots of a resistant line compared with a susceptible line. Preinoculation temperature did not modify resistance expression to later infections by root-knot nematodes. However, postinoculation temperatures affected development of Meloidogyne spp. in both the resistant and susceptible bean lines tested. The more rapid development of nematodes to adults at the higher postinoculation temperature of 28 °C in both bean lines suggests direct temperature effects on nematode development instead of on resistance expression of either of two gene systems. Also, resistance was stable at 30 °C and 32 °C.     

Biological mechanisms of aging predict age‐related disease co‐occurrence in patients

Genetic, environmental, and pharmacological interventions into the aging process can confer resistance to multiple age‐related diseases in laboratory animals, including rhesus monkeys. These findings imply that individual mechanisms of aging might contribute to the co‐occurrence of age‐related diseases in humans and could be targeted to prevent these conditions simultaneously. To address this question, we text mined 917,645 literature abstracts followed by manual curation and found strong, non‐random associations between age‐related diseases and aging mechanisms in humans, confirmed by gene set enrichment analysis of GWAS data. Integration of these associations with clinical data from 3.01 million patients showed that age‐related diseases associated with each of five aging mechanisms were more likely than chance to be present together in patients. Genetic evidence revealed that innate and adaptive immunity, the intrinsic apoptotic signaling pathway and activity of the ERK1/2 pathway were associated with multiple aging mechanisms and diverse age‐related diseases. Mechanisms of aging hence contribute both together and individually to age‐related disease co‐occurrence in humans and could potentially be targeted accordingly to prevent multimorbidity.     

Habitat‐linked genetic structure for white‐crowned sparrow (Zonotrichia leucophrys): Local factors shape population genetic structure

Ecological, environmental, and geographic factors all influence genetic structure. Species with broad distributions are ideal systems because they cover a range of ecological and environmental conditions allowing us to test which components predict genetic structure. This study presents a novel, broad geographic approach using molecular markers, morphology, and habitat modeling to investigate rangewide and local barriers causing contemporary genetic differentiation within the geographical range of three white‐crowned sparrow (Zonotrichia leucophrys) subspecies: Z. l. gambelii, Z. l. oriantha, and Z. l. pugetensis. Three types of genetic markers showed geographic distance between sampling sites, elevation, and ecosystem type are key factors contributing to population genetic structure. Microsatellite markers revealed white‐crowned sparrows do not group by subspecies, but instead indicated four groupings at a rangewide scale and two groupings based on coniferous and deciduous ecosystems at a local scale. Our analyses of morphological variation also revealed habitat differences; sparrows from deciduous ecosystems are larger than individuals from coniferous ecosystems based on principal component analyses. Habitat modeling showed isolation by distance was prevalent in describing genetic structure, but isolation by resistance also had a small but significant influence. Not only do these findings have implications concerning the accuracy of subspecies delineations, they also highlight the critical role of local factors such as habitat in shaping contemporary population genetic structure of species with high dispersal ability.     

Galangin as a direct inhibitor of vWbp protects mice from Staphylococcus aureus‐induced pneumonia

The surge in multidrug resistance in Staphylococcus aureus (S. aureus) and the lag in antibiotic discovery necessitate the development of new anti‐infective strategies to reduce S. aureus infections. In S. aureus, von Willebrand factor‐binding protein (vWbp) is not only the main coagulase that triggers host prothrombin activation and formation of fibrin cables but also bridges the bacterial cell wall and von Willebrand factor, thereby allowing S. aureus to bind to platelets and endothelial cells, playing a vital role in pathogenesis of S. aureus infections. Here, we have identified that galangin, a bioactive compound found in honey and Alpinia officinarum Hance, is a potent and direct inhibitor of vWbp by coagulation activity inhibition assay, thermal shift assay and biolayer interferometry assay. Molecular dynamic simulations and verification experiments revealed that the Trp‐64 and Leu‐69 residues are necessary for the binding of galangin to vWbp. Significantly, galangin attenuated S. aureus virulence in a mouse S. aureus‐induced pneumonia model. In addition, we also identified that galangin can enhance the therapeutic effect of latamoxef on S. aureus‐induced pneumonia. Taken together, the results suggest that galangin may be used for the development of therapeutic drugs or utilized as adjuvants to combine with antibiotics to combat S. aureus‐related infections.     

In-silico interaction studies suggest RND efflux pump mediates polymyxin resistance in Acinetobacter baumannii

Bacterial efflux pumps have emerged as antibiotic resistance determinants and confers multi-drug resistance to a broad range of antimicrobials as well as non-antibiotic substances. A study about translocation of antibiotic molecules through the efflux transporter, will contribute in determining substrate specificity. In the present study, we have explored RND family efflux pump extensively found in Acinetobacter baumannii i.e. AdeABC. Besides, another well studied RND efflux pump, AcrAB-TolC together with a non-RND efflux pump, NorM was investigated for comparative analysis. We employed a series of computational techniques ranging from molecular docking to binding free energy estimation and molecular dynamics simulations to determine the binding affinity for different classes of drugs, namely aminoglycosides, polymyxins, β-lactams, tetracyclines, glycylcyclines, quinolones and metronidazole with AdeB, AcrB, and NorM efflux proteins. Our results revealed that class polymyxins has the highest binding affinity with the RND efflux pumps i.e. AcrAB-TolC and AdeABC as well as non-RND efflux pump, NorM. The experimental validation study demonstrated bigger zone of inhibition in presence of efflux pump inhibitor than polymyxin alone thus unveiling its specificity toward efflux pump. The reported experimental data comprising of minimum inhibitory concentration of antibiotics toward these efflux pumps also support our finding based on in silico approach. To recapitulate the outcome, polymyxins shows maximum specificity toward RND as well as non-RND efflux pump and may unlatch the way to rationally develop new potential antibacterial agents as well as efflux pump inhibitors in order to combat resistance.     

Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil

New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the development of new and innovative methods of mosquito control based around the Sterile Insect Technique (SIT)^1-3. A control method known as RIDL (Release of Insects carrying a Dominant Lethal)⁴, is based around SIT, but uses genetic methods to remove the need for radiation-sterilization^5-8. A RIDL strain of Ae. aegypti was successfully tested in the field in Grand Cayman^9,10; further field use is planned or in progress in other countries around the world.Mass rearing of insects has been established in several insect species and to levels of billions a week. However, in mosquitoes, rearing has generally been performed on a much smaller scale, with most large scale rearing being performed in the 1970s and 80s. For a RIDL program it is desirable to release as few females as possible as they bite and transmit disease. In a mass rearing program there are several stages to produce the males to be released: egg production, rearing eggs until pupation, and then sorting males from females before release. These males are then used for a RIDL control program, released as either pupae or adults^11,12.To suppress a mosquito population using RIDL a large number of high quality male adults need to be reared^13,14. The following describes the methods for the mass rearing of OX513A, a RIDL strain of Ae. aegypti ⁸, for release and covers the techniques required for the production of eggs and mass rearing RIDL males for a control program.