Randomly barcoded transposon mutant libraries for gut commensals I: Strategies for efficient library construction.

Randomly barcoded transposon mutant libraries are powerful tools for studying gene function and organization, assessing gene essentiality and pathways, discovering potential therapeutic targets, and understanding the physiology of gut bacteria and their interactions with the host. However, construction of high-quality libraries with uniform representation can be challenging. In this review, we survey various strategies for barcoded library construction, including transposition systems, methods of transposon delivery, optimal library size, and transconjugant selection schemes. We discuss the advantages and limitations of each approach, as well as factors to consider when selecting a strategy. In addition, we highlight experimental and computational advances in arraying condensed libraries from mutant pools. We focus on examples of successful library construction in gut bacteria and their application to gene function studies and drug discovery. Given the need for understanding gene function and organization in gut bacteria, we provide a comprehensive guide for researchers to construct randomly barcoded transposon mutant libraries

Defining a spinal microcircuit that gates myelinated afferent input: implications for tactile allodynia

Chronic pain presents a major unmet clinical problem. The development of more effective treatments is hindered by our limited understanding of the neuronal circuits underlying sensory perception. Here, we show that parvalbumin (PV)-expressing dorsal horn interneurons modulate the passage of sensory information conveyed by low-threshold mechanoreceptors (LTMRs) directly via presynaptic inhibition and also gate the polysynaptic relay of LTMR input to pain circuits by inhibiting lamina II excitatory interneurons whose axons project into lamina I. We show changes in the functional properties of these PV interneurons following peripheral nerve injury and that silencing these cells unmasks a circuit that allows innocuous touch inputs to activate pain circuits by increasing network activity in laminae I–IV. Such changes are likely to result in the development of tactile allodynia and could be targeted for more effective treatment of mechanical pain

E-cadherin transcriptional downregulation by promoter methylation but not mutation is related to epithelial-to-mesenchymal transition in breast cancer cell lines

Using genome-wide expression profiling of a panel of 27 human mammary cell lines with different mechanisms of E-cadherin inactivation, we evaluated the relationship between E-cadherin status and gene expression levels. Expression profiles of cell lines with E-cadherin (CDH1) promoter methylation were significantly different from those with CDH1 expression or, surprisingly, those with CDH1 truncating mutations. Furthermore, we found no significant differentially expressed genes between cell lines with wild-type and mutated CDH1. The expression profile complied with the fibroblastic morphology of the cell lines with promoter methylation, suggestive of epithelial-mesenchymal transition (EMT). All other lines, also the cases with CDH1 mutations, had epithelial features. Three non-tumorigenic mammary cell lines derived from normal breast epithelium also showed CDH1 promoter methylation, a fibroblastic phenotype and expression profile. We suggest that CDH1 promoter methylation, but not mutational inactivation, is part of an entire programme, resulting in EMT and increased invasiveness in breast cancer. The molecular events that are part of this programme can be inferred from the differentially expressed genes and include genes from the TGFbeta pathway, transcription factors involved in CDH1 regulation (i.e. ZFHX1B, SNAI2, but not SNAI1, TWIST), annexins, AP1/2 transcription factors and members of the actin and intermediate filament cytoskeleton organisation.Toxicolog

Microwave determination of the quasiparticle scattering time in YBa2Cu3O6.95

We report microwave surface resistance (Rs) measurements on two very-high-quality YBa2Cu3O6.95 crystals which exhibit extremely low residual loss at 1.2 K (2-6 μΩ at 2 GHz), a broad, reproducible peak at around 38 K, and a rapid increase in loss, by 4 orders of magnitude, between 80 and 93 K. These data provide one ingredient in the determination of the temperature dependence of the real part of the microwave conductivity, σ1(T), and of the quasiparticle scattering time. The other necessary ingredient is an accurate knowledge of the magnitude and temperature dependence of the London penetration depth, λ(T). This is derived from published data, from microwave data of Anlage, Langley, and co-workers and from, high-quality μSR data. We infer, from a careful analysis of all available data, that λ2(0)/λ2(T) is well approximated by the simple function 1-t2, where t=T/Tc, and that the low-temperature data are incompatible with the existence of an s-wave, BCS-like gap. Combining the Rs and λ(T) data, we find that σ1(T), has a broad peak around 32 K with a value about 20 times that at Tc. Using a generalized two-fluid model, we extract the temperature dependence of the quasiparticle scattering rate which follows an exponential law, exp(T/T0), where T0≊12 K, for T between 15 and 84 K. Such a temperature dependence has previously been observed in measurements of the nuclear spin-lattice relaxation rate. Both the uncertainties in our analysis and the implications for the mechanism of high-temperature superconductivity are discussed

Dual-Pharmacophore Pyrithione-Containing Cephalosporins Kill Both Replicating and Nonreplicating Mycobacterium tuberculosis

The historical view of β-lactams as ineffective antimycobacterials has given way to growing interest in the activity of this class against Mycobacterium tuberculosis (Mtb) in the presence of a β-lactamase inhibitor. However, most antimycobacterial β-lactams kill Mtb only or best when the bacilli are replicating. Here, a screen of 1904 β-lactams led to the identification of cephalosporins substituted with a pyrithione moiety at C3′ that are active against Mtb under both replicating and nonreplicating conditions, neither activity requiring a β-lactamase inhibitor. Studies showed that activity against nonreplicating Mtb required the in situ release of the pyrithione, independent of the known class A β-lactamase, BlaC. In contrast, replicating Mtb could be killed both by released pyrithione and by the parent β-lactam. Thus, the antimycobacterial activity of pyrithione-containing cephalosporins arises from two mechanisms that kill mycobacteria in different metabolic states