Principles and practice of determining metal–protein affinities

Metal ions play many critical roles in biology, as structural and catalytic cofactors, and as cell regulatory and signalling elements. The metal–protein affinity, expressed conveniently by the metal dissociation constant, KD, describes the thermodynamic strength of a metal–protein interaction and is a key parameter that can be used, for example, to understand how proteins may acquire metals in a cell and to identify dynamic elements (e.g. cofactor binding, changing metal availabilities) which regulate protein metalation in vivo. Here, we outline the fundamental principles and practical considerations that are key to the reliable quantification of metal–protein affinities. We review a selection of spectroscopic probes which can be used to determine protein affinities for essential biological transition metals (including Mn(II), Fe(II), Co(II), Ni(II), Cu(I), Cu(II) and Zn(II)) and, using selected examples, demonstrate how rational probe selection combined with prudent experimental design can be applied to determine accurate KD values.

Ribo-Pop: Simple, cost-effective, and widely applicable ribosomal RNA depletion

ABSTRACTThe measurement of RNA abundance derived from massively parallel sequencing experiments is an essential technique. Methods that reduce ribosomal RNA levels are usually required prior to sequencing library construction because ribosomal RNA typically comprises the vast majority of a total RNA sample. For some experiments, ribosomal RNA depletion is favored over poly(A) selection because it offers a more inclusive representation of the transcriptome. However, methods to deplete ribosomal RNA are generally proprietary, complex, inefficient, applicable to only specific species, or compatible with only a narrow range of RNA input levels. Here, we describe Ribo-Pop (ribosomal RNA depletion for popular use), a simple workflow and antisense oligo design strategy that we demonstrate works over a wide input range and can be easily adapted to any organism with a sequenced genome. We provide a computational pipeline for probe selection, a streamlined 20-minute protocol, and ready-to-use oligo sequences for several organisms. We anticipate that our simple and generalizable “open source” design strategy would enable virtually any lab to pursue full transcriptome sequencing in their organism of interest with minimal time and resource investment.

In Vivo Reflectance Confocal Microscopy in General Dermatology: How to Choose the Right Indication

Reflectance confocal microscopy (RCM) is a high-resolution, noninvasive imaging technique being increasingly used as an aid to diagnosis in the dermatology setting. RCM is applied in the diagnosis of both melanoma and nonmelanoma skin tumors, but also in the interpretation and management of inflammatory skin diseases. Two different devices with different designs for specific indications are available in the market: a static and a handheld probe. Several clinical presentations of the lesion could affect the examination, such as the presence of ulceration or hyperkeratosis; moreover, the anatomical site can drive the probe selection as well as the effective indication to RCM examination. In this review article, indications for the use of RCM are described in detail with a schematic approach for practical purposes.