Mis-spliced transcripts generate de novo proteins in TDP-43-related ALS/FTD.

Functional loss of TDP-43, an RNA binding protein genetically and pathologically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), leads to the inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote the degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. Here, we show that mRNA transcripts harboring cryptic exons generated de novo proteins in TDP-43-depleted human iPSC-derived neurons in vitro, and de novo peptides were found in cerebrospinal fluid (CSF) samples from patients with ALS or FTD. Using coordinated transcriptomic and proteomic studies of TDP-43-depleted human iPSC-derived neurons, we identified 65 peptides that mapped to 12 cryptic exons. Cryptic exons identified in TDP-43-depleted human iPSC-derived neurons were predictive of cryptic exons expressed in postmortem brain tissue from patients with TDP-43 proteinopathy. These cryptic exons produced transcript variants that generated de novo proteins. We found that the inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Last, we showed that 18 de novo peptides across 13 genes were present in CSF samples from patients with ALS/FTD spectrum disorders. The demonstration of cryptic exon translation suggests new mechanisms for ALS/FTD pathophysiology downstream of TDP-43 dysfunction and may provide a potential strategy to assay TDP-43 function in patient CSF.

Mis-spliced transcripts generate de novo proteins in TDP-43-related ALS/FTD.

Functional loss of TDP-43, an RNA-binding protein genetically and pathologically linked to ALS and FTD, leads to inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. However, the possibility of de novo protein synthesis from cryptic exon transcripts has not been explored. Here, we show that mRNA transcripts harboring cryptic exons generate de novo proteins both in TDP-43 deficient cellular models and in disease. Using coordinated transcriptomic and proteomic studies of TDP-43 depleted iPSC-derived neurons, we identified numerous peptides that mapped to cryptic exons. Cryptic exons identified in iPSC models were highly predictive of cryptic exons expressed in brains of patients with TDP-43 proteinopathy, including cryptic transcripts that generated de novo proteins. We discovered that inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Finally, we showed that these de novo peptides were present in CSF from patients with ALS. The demonstration of cryptic exon translation suggests new mechanisms for ALS pathophysiology downstream of TDP-43 dysfunction and may provide a strategy for novel biomarker development.

A reference human induced pluripotent stem cell line for large-scale collaborative studies.

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field.

Raman imaging.

The past decade has seen an enormous increase in the number and breadth of imaging techniques developed for analysis in many industries, including pharmaceuticals, food, and especially biomedicine. Rather than accept single-dimensional forms of information, users now demand multidimensional assessment of samples. High specificity and the need for little or no sample preparation make Raman imaging a highly attractive analytical technique and provide motivation for continuing advances in its supporting technology and utilization. This review discusses the current tools employed in Raman imaging, the recent advances, and the major applications in this ever-growing analytical field.

Waterborne pathogen detection using Raman spectroscopy.

Raman spectroscopy is being evaluated as a candidate technology for waterborne pathogen detection. We have investigated the impact of key experimental and background interference parameters on the bacterial species level identification performance of Raman detection. These parameters include laser-induced photodamage threshold, composition of water matrix, and organism aging in water. The laser-induced photodamage may be minimized by operating a 532 nm continuous wave laser excitation at laser power densities below 2300 W/cm(2) for Grampositive Bacillus atrophaeus (formerly Bacillus globigii, BG) vegetative cells, 2800 W/cm(2) for BG spores, and 3500 W/cm(2) for Gram-negative E. coli (EC) organisms. In general, Bacillus spore microorganism preparations may be irradiated with higher laser power densities than the equivalent Bacillus vegetative preparations. In order to evaluate the impact of background interference and organism aging, we selected a biomaterials set comprising Gram-positive (anthrax simulants) organisms, Gram-negative (plague simulant) organisms, and proteins (toxin simulants) and constructed a Raman signature classifier that identifies at the species level. Subsequently, we evaluated the impact of tap water and storage time in water (aging) on the classifier performance when characterizing B. thuringiensis spores, BG spores, and EC cell preparations. In general, the measured Raman signatures of biological organisms exhibited minimal spectral variability with respect to the age of a resting suspension and water matrix composition. The observed signature variability did not substantially degrade discrimination performance at the genus and species levels. In addition, Raman chemical imaging spectroscopy was used to distinguish a mixture of BG spores and EC cells at the single cell level.

Raman chemical imaging for ingredient-specific particle size characterization of aqueous suspension nasal spray formulations: a progress report.

PURPOSE: This study was conducted to evaluate the feasibility of using Raman chemical imaging (i.e., Raman imaging microspectroscopy) to establish chemical identity, particle size and particle size distribution (PSD) for a representative corticosteroid in aqueous nasal spray suspension formulations. MATERIALS AND METHODS: The Raman imaging PSD protocol was validated using polystyrene (PS) microsphere size standards (NIST-traceable). A Raman spectral library was developed for the active and inactive compounds in the formulation. Four nasal sprays formulated with beclomethasone dipropionate (BDP) ranging in size from 1.4 to 8.3 microm were imaged by both Raman and brightfield techniques. The Raman images were then processed to calculate the PSD for each formulation. RESULTS: Within each region examined, active pharmaceutical ingredient (API) particles are unambiguously identified and the total number of those particles, particle size and PSD of API free of excipients and PSD of API particles adhered to other excipients are reported. CONCLUSIONS: Good statistical agreement is obtained between the reported and measured sizes of the PS microspheres. BDP particles were clearly distinguishable from those of excipients. Raman chemical imaging (RCI) is able to differentiate between and identify the chemical makeup of multiple components in complex BDP sample and placebo mixtures. The Raman chemical imaging method (coupled Raman and optical imaging) shows promise as a method for characterizing particle size and shape of corticosteroid in aqueous nasal spray suspension formulations. However, rigorous validation of RCI for PSD analysis is incomplete and requires additional research effort. Some specific areas of concern are discussed.

Raman chemical imaging spectroscopy reagentless detection and identification of pathogens: signature development and evaluation.

An optical detection method, Raman chemical imaging spectroscopy (RCIS), is reported, which combines Raman spectroscopy, fluorescence spectroscopy, and digital imaging. Using this method, trace levels of biothreat organisms are detected in the presence of complex environmental backgrounds without the use of amplification or enhancement techniques. RCIS is reliant upon the use of Raman signatures and automated recognition algorithms to perform species-level identification. The rationale and steps for constructing a pathogen Raman signature library are described, as well as the first reported Raman spectra from live, priority pathogens, including Bacillus anthracis, Yersinia pestis, Burkholderia mallei, Francisella tularensis, Brucella abortus, and ricin. Results from a government-managed blind trial evaluation of the signature library demonstrated excellent specificity under controlled laboratory conditions.

Forensic applications of chemical imaging: latent fingerprint detection using visible absorption and luminescence.

Chemical imaging technology is a rapid examination technique that combines molecular spectroscopy and digital imaging, providing information on morphology, composition, structure, and concentration of a material. Among many other applications, chemical imaging offers an array of novel analytical testing methods, which limits sample preparation and provides high-quality imaging data essential in the detection of latent fingerprints. Luminescence chemical imaging and visible absorbance chemical imaging have been successfully applied to ninhydrin, DFO, cyanoacrylate, and luminescent dye-treated latent fingerprints, demonstrating the potential of this technology to aid forensic investigations. In addition, visible absorption chemical imaging has been applied successfully to visualize untreated latent fingerprints.