Loci Encoding Compounds Potentially Active Against Drug-Resistant Pathogens amidst a Decreasing Pool of Novel Antibiotics

Since the discovery of penicillin, microbes have been a source of antibiotics that inhibit the growth of pathogens. However, with the evolution of multidrug-resistant (MDR) strains, it remains unclear if there is an abundant or limited supply of natural products to be discovered that are effective against MDR isolates. To identify strains that are antagonistic to pathogens, we examined a set of 471 globally derived environmental strains (env-Ps) for activity against a panel of 65 pathogens including spp., spp., , and spp. isolated from the lungs of cystic fibrosis (CF) patients. From more than 30,000 competitive interactions, 1,530 individual inhibitory events were observed. While strains from water habitats were not proportionate in antagonistic activity, MDR CF-derived pathogens (CF-Ps) were less susceptible to inhibition by env-Ps, suggesting that fewer natural products are effective against MDR strains. These results advocate for a directed strategy to identify unique drugs. To facilitate discovery of antibiotics against the most resistant pathogens, we developed a workflow in which phylogenetic and antagonistic data were merged to identify strains that inhibit MDR CF-Ps and subjected those env-Ps to transposon mutagenesis. Six different biosynthetic gene clusters (BGCs) were identified from four strains whose products inhibited pathogens including carbapenem-resistant BGCs were rare in databases, suggesting the production of novel antibiotics. This strategy can be utilized to facilitate the discovery of needed antibiotics that are potentially active against the most drug-resistant pathogens. Carbapenem-resistant is difficult to treat and has been deemed by the World Health Organization as a priority one pathogen for which antibiotics are most urgently needed. Although metagenomics and bioinformatic studies suggest that natural bacteria remain a source of novel compounds, the identification of genes and their products specific to activity against MDR pathogens remains problematic. Here, we examine water-derived pseudomonads and identify gene clusters whose compounds inhibit CF-derived MDR pathogens, including carbapenem-resistant

Environmental pseudomonads inhibit cystic fibrosis patient-derived Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen which is evolving resistance to many currently used antibiotics. While much research has been devoted to the roles of pathogenic P. aeruginosa in cystic fibrosis (CF) patients, less is known of its ecological properties. P. aeruginosa dominates the lungs during chronic infection in CF patients, yet its abundance in some environments is less than that of other diverse groups of pseudomonads. Here, we sought to determine if clinical isolates of P. aeruginosa are vulnerable to environmental pseudomonads that dominate soil and water habitats in one-to-one competitions which may provide a source of inhibitory factors. We isolated a total of 330 pseudomonads from diverse habitats of soil and freshwater ecosystems and competed these strains against one another to determine their capacity for antagonistic activity. Over 900 individual inhibitory events were observed. Extending the analysis to P. aeruginosa isolates revealed that clinical isolates, including ones with increased alginate production, were susceptible to competition by multiple environmental strains. We performed transposon mutagenesis on one isolate and identified an ~14.8-kb locus involved in antagonistic activity. Only two other environmental isolates were observed to carry the locus, suggesting the presence of additional unique compounds or interactions among other isolates involved in outcompeting P. aeruginosa. This collection of strains represents a source of compounds that are active against multiple pathogenic strains. With the evolution of resistance of P. aeruginosa to currently used antibiotics, these environmental strains provide opportunities for novel compound discovery against drug-resistant clinical strains

Immune Differentiation Regulator p100 Tunes NF-κB Responses to TNF

Tumor necrosis factor (TNF) is a pleiotropic cytokine whose primary physiological function involves coordinating inflammatory and adaptive immune responses. However, uncontrolled TNF signaling causes aberrant inflammation and has been implicated in several human ailments. Therefore, an understanding of the molecular mechanisms underlying dynamical and gene controls of TNF signaling bear significance for human health. As such, TNF engages the canonical nuclear factor kappa B (NF-κB) pathway to activate RelA:p50 heterodimers, which induce expression of specific immune response genes. Brief and chronic TNF stimulation produces transient and long-lasting NF-κB activities, respectively. Negative feedback regulators of the canonical pathway, including IκBα, are thought to ensure transient RelA:p50 responses to short-lived TNF signals. The non-canonical NF-κB pathway mediates RelB activity during immune differentiation involving p100. We uncovered an unexpected role of p100 in TNF signaling. Brief TNF stimulation of p100-deficient cells triggered an additional late NF-κB activity consisting of RelB:p50 heterodimers, which modified the TNF-induced gene-expression program. In p100-deficient cells subjected to brief TNF stimulation, RelB:p50 not only sustained the expression of a subset of RelA-target immune response genes but also activated additional genes that were not normally induced by TNF in WT mouse embryonic fibroblasts (MEFs) and were related to immune differentiation and metabolic processes. Despite this RelB-mediated distinct gene control, however, RelA and RelB bound to mostly overlapping chromatin sites in p100-deficient cells. Repeated TNF pulses strengthened this RelB:p50 activity, which was supported by NF-κB-driven RelB synthesis. Finally, brief TNF stimulation elicited late-acting expressions of NF-κB target pro-survival genes in p100-deficient myeloma cells. In sum, our study suggests that the immune-differentiation regulator p100 enforces specificity of TNF signaling and that varied p100 levels may provide for modifying TNF responses in diverse physiological and pathological settings

Abnormal Blink Reflex and Intermuscular Coherence in Writer's Cramp

Background: Writer's cramp (WC) is a task-specific focal hand dystonia presenting with pain, stiffness and/or tremor while writing. We explored the involvement of cortical and brainstem circuits by measuring intermuscular coherence (IMC) and pre-pulse inhibition (PPI) of the blink reflex.Methods: IMC was measured in 10 healthy controls and 20 WC patients (10 with associated tremor) while they performed a precision grip task at different force levels. Blink responses were evaluated in 9 healthy controls and 10 WC patients by stimulating the right supraorbital nerve and recording surface EMG from the orbicularis oculi muscles bilaterally. PPI involved conditioning this stimulation with a prior shock to the right median nerve (100 ms interval), and measuring the reduction in the R2 component of the blink reflex.Results: Significant IMC at 3–7 Hz was present in WC patients, but not in healthy controls. Compared to healthy controls, in WC patients the R2 component of the blink reflex showed significantly less PPI. IMC at 3–7 Hz could reliably discriminate WC patients from healthy controls.Conclusion: Cortical or sub-cortical circuits generating theta (3–7 Hz) oscillations might play an important role in the pathogenesis of WC. Moreover, the lack of PPI implicates abnormalities in brainstem inhibition in the emergence of WC. IMC may merit further development as an electrodiagnostic test for focal dystonia

Disparity in the DNA translocase domains of SWI/SNF and ISW2

An ATP-dependent DNA translocase domain consisting of seven conserved motifs is a general feature of all ATP-dependent chromatin remodelers. While motifs on the ATPase domains of the yeast SWI/SNF and ISWI families of remodelers are highly conserved, the ATPase domains of these complexes appear not to be functionally interchangeable. We found one reason that may account for this is the ATPase domains interact differently with nucleosomes even though both associate with nucleosomal DNA 17–18 bp from the dyad axis. The cleft formed between the two lobes of the ISW2 ATPase domain is bound to nucleosomal DNA and Isw2 associates with the side of nucleosomal DNA away from the histone octamer. The ATPase domain of SWI/SNF binds to the same region of nucleosomal DNA, but is bound outside of the cleft region. The catalytic subunit of SWI/SNF also appears to intercalate between the DNA gyre and histone octamer. The altered interactions of SWI/SNF with DNA are specific to nucleosomes and do not occur with free DNA. These differences are likely mediated through interactions with the histone surface. The placement of SWI/SNF between the octamer and DNA could make it easier to disrupt histone–DNA interactions

Environmental Pseudomonas are a source of Novel Antibiotics that inhibit Cystic fibrosis derived pathogenic Pseudomonas aeruginosa

The emergence of antimicrobial resistance bacteria has become a major threat to human society. The rapid spread of resistant pathogens and the associated loss of efficacy of available drugs needs to be met with the development of antibiotics and alternative treatments. Pseudomonas aeruginosa is an opportunistic human pathogen evolving resistance to many currently used antibiotics. Chronic lung infections with the bacterium P. aeruginosa are the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. Escalating this problem is that pharmaceutical companies have dropped drug development due to low profitability, thus making the efforts of drug discovery of prime importance. To address this global health threat research institutes have now stepped forward to aid in discovery of novel compounds. P. aeruginosa dominates the lungs during chronic infections in CF patients, yet it’s abundance in non-human habitats such as water and soil is less compared to other diverse groups of pseudomonads. A trait that could contribute to such decreased abundance is bacterial competition from other Pseudomonas populations that dominate water and soil habitats. We hypothesized that environmental Pseudomonas from diverse soil and water habitats produce secondary metabolites capable of inhibiting the growth of CF derived P. aeruginosa. Here, we sought to determine if clinical isolates of P. aeruginosa are susceptible to competition by environmental pseudomonads which may provide a source of inhibitory factors. We have used a population based study in association with transposon mutagenesis, PCR techniques, whole genome sequencing and bioinformatic analysis to identify environmental Pseudomonas biosynthetic gene clusters (BGCs) and characterize antagonistic compounds that are effective against CF-derived P. aeruginosa. A total of five BGCs have been identified in this study from environmental Pseudomonas strains S4B6, S3F9 (soil-derived) and LE6C6 (water-derived) encoding diverse compounds such as bacteriocins, NRPSs, phenazines, and siderophores involved in antagonistic activity. Extending this analysis, we have also identified environmental Pseudomonas that inhibit not only CF-derived P. aeruginosa but are effective against other pathogens including ESKAPE pathogens and carbapenem resistant P. aeruginosa. Overall, this research serves as a platform for the identification of novel antibiotics from these environmental isolates

Environmental Pseudomonas are a source of Novel Antibiotics that inhibit Cystic fibrosis derived pathogenic Pseudomonas aeruginosa

The emergence of antimicrobial resistance bacteria has become a major threat to human society. The rapid spread of resistant pathogens and the associated loss of efficacy of available drugs needs to be met with the development of antibiotics and alternative treatments. Pseudomonas aeruginosa is an opportunistic human pathogen evolving resistance to many currently used antibiotics. Chronic lung infections with the bacterium P. aeruginosa are the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. Escalating this problem is that pharmaceutical companies have dropped drug development due to low profitability, thus making the efforts of drug discovery of prime importance. To address this global health threat research institutes have now stepped forward to aid in discovery of novel compounds. P. aeruginosa dominates the lungs during chronic infections in CF patients, yet it’s abundance in non-human habitats such as water and soil is less compared to other diverse groups of pseudomonads. A trait that could contribute to such decreased abundance is bacterial competition from other Pseudomonas populations that dominate water and soil habitats. We hypothesized that environmental Pseudomonas from diverse soil and water habitats produce secondary metabolites capable of inhibiting the growth of CF derived P. aeruginosa. Here, we sought to determine if clinical isolates of P. aeruginosa are susceptible to competition by environmental pseudomonads which may provide a source of inhibitory factors. We have used a population based study in association with transposon mutagenesis, PCR techniques, whole genome sequencing and bioinformatic analysis to identify environmental Pseudomonas biosynthetic gene clusters (BGCs) and characterize antagonistic compounds that are effective against CF-derived P. aeruginosa. A total of five BGCs have been identified in this study from environmental Pseudomonas strains S4B6, S3F9 (soil-derived) and LE6C6 (water-derived) encoding diverse compounds such as bacteriocins, NRPSs, phenazines, and siderophores involved in antagonistic activity. Extending this analysis, we have also identified environmental Pseudomonas that inhibit not only CF-derived P. aeruginosa but are effective against other pathogens including ESKAPE pathogens and carbapenem resistant P. aeruginosa. Overall, this research serves as a platform for the identification of novel antibiotics from these environmental isolates

Short-term changes in axial length during simulations of typical far, intermediate and near tasks [Conference abstract]

Purpose: To investigate the changes in axial length with the combined effect of accommodation and angle of gaze (convergence and downward gaze) over 5 minutes in groups of myopes and emmetropes. Methods: A total of 31 subjects (nine emmetropes, 10 low myopes, and 12 moderate to high myopes) aged from 18 to 31 years were recruited. To measure ocular biometrics in inferonasal gaze with accommodation, an optical biometer (Lenstar LS900) was inclined on a tilt and height adjustable stage, with the subject’s chinrest mounted on a rotary stage to induce various levels of convergence by rotation of the subject’s head in primary or downward gaze. Initially, the subjects performed a distance viewing task in primary gaze for 10 minutes to provide a ‘wash-out’ period for prior visual tasks, and then the subject’s axial length and ocular biometrics were measured in nine different combinations of gaze/accommodation over 5 minutes. These nine sessions for all gaze measurements (i.e. three levels of accommodation 9 three levels of convergence) were completed across 3 days of testing (one accommodation condition on each day).The nine combinations of gaze/accommodation were based on those required to view the centre, right and left edges of a distant TV at 6 m in primary gaze, an intermediate task (i.e. computer at 50 cm in 10° downward gaze) and a near task (i.e. reading A4 page at 20 cm in 20° downward gaze). Subjects were wearing a custom built three-axes head tracker throughout the experiment that monitored subjects’ relative head movements (roll, pitch and yaw) during measurements. Results: A significant increase in axial length occurred with the combined effect of accommodation, convergence and downward gaze (repeated measures ANOVA, p 0.05). Moderate to high myopes had a greater change in the axial length (mean change 40 ± 11 lm after 5 minutes of near task) than that of emmetropes (mean change 29 ± 15 lm after 5 minutes of near task) and low myopes (mean change 29 ± 16 lm after 5 minutes of near task) associated with time (p = 0.02) and accommodation by time (p = 0.03). Conclusions: The combination of accommodation, convergence and downward angle has a significant short term effect on axial length over time. The near task in downward gaze with convergence caused a greater change in axial length than the intermediate and distant visual tasks. The greater axial elongation measured in the infero-nasal direction with accommodation is most likely associated with a combination of biomechanical factors such as, extraocular muscle forces and ciliary muscle contraction

Axial elongation associated with biomechanical factors during near work

Purpose: To investigate the changes occurring in the axial length, choroidal thickness and anterior biometrics of the eye during a 10 minute near task performed in downward gaze. Methods: Twenty young adult subjects (10 emmetropes and 10 myopes) participated in this study. To measure ocular biometrics in downward gaze, an optical biometer was inclined on a custom built, height and tilt adjustable table. Baseline measures were collected after each subject performed a distance primary gaze control task for 10 mins, to provide wash-out period for prior visual tasks before each of three different accommodation/gaze conditions. These other three conditions included a near task (2.5 D) in primary gaze, and a near (2.5 D) and a far (0 D) accommodative task in downward gaze (25°), all for 10 mins duration. Immediately after, and then 5 and 10 mins from the commencement of each trial, measurements of ocular biometrics (e.g. anterior biometrics, axial length, choroidal thickness and retinal thickness) were obtained. Results: Axial length increased with accommodation and was significantly greater for downward gaze with accommodation (mean change ± SD 23 ± 13 µm at 10 mins) compared to primary gaze with accommodation (mean change 8 ± 15 µm at 10 mins) (p < 0.05). A small amount of choroidal thinning was also found during accommodation that was statistically significant in downward gaze (13 ± 14 µm at 10 mins, p < 0.05). Accommodation in downward gaze also caused greater changes in anterior chamber depth and lens thickness compared to accommodation in primary gaze. Conclusion: Axial length, choroidal thickness and anterior eye biometrics change significantly during accommodation in downward gaze as a function of time. These changes appear to be due to the combined influence of biomechanical factors (i.e. extraocular muscle forces, ciliary muscle contraction) associated with near tasks in downward gaze

Surface Morphology and Exchange Bias Anisotropy Studies in Large Area Deposited Co2FeSi Ir50Mn50 Multi Layers For Spintronic Applications

Surface morphology and magnetic properties of ferromagnetic Heusler alloy Co2FeSi thin films and their multi-layers with anti-ferromagnetic Ir50Mn50, which find applications in spintronic devices were investigated. The sputtering process flow for large area deposition of thin films on 3 inch size thermally oxidized single crystal Si(100)/SiO2 substrates have been developed by optimizing the sputtering geometry and other process parameters. A uniform film composition, thickness, smooth surface, good crystallinity and magnetic properties have been achieved in the films over 3-inch size wafers. The isotropic magnetic properties such as saturation/remanent magnetizations, coercivity were achieved in Co2FeSi films deposited on 3-inch size Si(100)/SiO2 wafers with 15 nm Cr buffer layer. An exchange bias anisotropy has been established in Co2FeSi/IrMn multilayer by magnetic annealing process using in-house made magnetic annealing set up. A maximum exchange bias anisotropy field, Hex of 178 Oe and low coercivity, Hc of 85 Oe has been achieved in the Co2FeSi/IrMn multilayer stacks suitable for magnetic tunnel junctions for spintronic applications