Discovery of a Substrate Selectivity Switch in Tyrosine Ammonia-Lyase, a Member of the Aromatic Amino Acid Lyase Family

SummaryTyrosine ammonia-lyase (TAL) is a recently described member of the aromatic amino acid lyase family, which also includes phenylalanine (PAL) and histidine ammonia-lyases (HAL). TAL is highly selective for L-tyrosine, and synthesizes 4-coumaric acid as a protein cofactor or antibiotic precursor in microorganisms. In this report, we identify a single active site residue important for substrate selection in this enzyme family. Replacing the active site residue His89 with Phe in TAL completely switched its substrate selectivity from tyrosine to phenylalanine, thereby converting it into a highly active PAL. When a corresponding mutation was made in PAL, the enzyme lost PAL activity and gained TAL activity. The discovered substrate selectivity switch is a rare example of a complete alteration of substrate specificity by a single point mutation. We also show that the identity of the amino acid at the switch position can serve as a guide to predict substrate specificities of annotated aromatic amino acid lyases in genome sequences

An influenza virus-triggered SUMO switch orchestrates co-opted endogenous retroviruses to stimulate host antiviral immunity

Dynamic small ubiquitin-like modifier (SUMO) linkages to diverse cellular protein groups are critical to orchestrate resolution of stresses such as genome damage, hypoxia, or proteotoxicity. Defense against pathogen insult (often reliant upon host recognition of "non-self" nucleic acids) is also modulated by SUMO, but the underlying mechanisms are incompletely understood. Here, we used quantitative SILAC-based proteomics to survey pan-viral host SUMOylation responses, creating a resource of almost 600 common and unique SUMO remodeling events that are mounted during influenza A and B virus infections, as well as during viral innate immune stimulation. Subsequent mechanistic profiling focused on a common infection-induced loss of the SUMO-modified form of TRIM28/KAP1, a host transcriptional repressor. By integrating knockout and reconstitution models with system-wide transcriptomics, we provide evidence that influenza virus-triggered loss of SUMO-modified TRIM28 leads to derepression of endogenous retroviral (ERV) elements, unmasking this cellular source of "self" double-stranded (ds)RNA. Consequently, loss of SUMO-modified TRIM28 potentiates canonical cytosolic dsRNA-activated IFN-mediated defenses that rely on RIG-I, MAVS, TBK1, and JAK1. Intriguingly, although wild-type influenza A virus robustly triggers this SUMO switch in TRIM28, the induction of IFN-stimulated genes is limited unless expression of the viral dsRNA-binding protein NS1 is abrogated. This may imply a viral strategy to antagonize such a host response by sequestration of induced immunostimulatory ERV dsRNAs. Overall, our data reveal that a key nuclear mechanism that normally prevents aberrant expression of ERV elements (ERVs) has been functionally co-opted via a stress-induced SUMO switch to augment antiviral immunity.</p

Latent regulatory potential of human-specific repetitive elements

At least half of the human genome is derived from repetitive elements, which are often lineage specific and silenced by a variety of genetic and epigenetic mechanisms. Using a transchromosomic mouse strain that transmits an almost complete single copy of human chromosome 21 via the female germline, we show that a heterologous regulatory environment can transcriptionally activate transposon-derived human regulatory regions. In the mouse nucleus, hundreds of locations on human chromosome 21 newly associate with activating histone modifications in both somatic and germline tissues, and influence the gene expression of nearby transcripts. These regions are enriched with primate and human lineage-specific transposable elements, and their activation corresponds to changes in DNA methylation at CpG dinucleotides. This study reveals the latent regulatory potential of the repetitive human genome and illustrates the species specificity of mechanisms that control it

High Harvest Yield, High Expansion, and Phenotype Stability of CD146 Mesenchymal Stromal Cells from Whole Primitive Human Umbilical Cord Tissue

Human umbilical cord blood is an excellent primitive source of noncontroversial stem cells for treatment of hematologic disorders; meanwhile, new stem cell candidates in the umbilical cord (UC) tissue could provide therapeutic cells for nonhematologic disorders. We show novel in situ characterization to identify and localize a panel of some markers expressed by mesenchymal stromal cells (MSCs; CD44, CD105, CD73, CD90) and CD146 in the UC. We describe enzymatic isolation and purification methods of different UC cell populations that do not require manual separation of the vessels and stroma of the coiled, helical-like UC tissue. Unique quantitation of in situ cell frequency and stromal cell counts upon harvest illustrate the potential to obtain high numerical yields with these methods. UC stromal cells can differentiate to the osteogenic and chondrogenic lineages and, under specific culturing conditions, they exhibit high expandability with unique long-term stability of their phenotype. The remarkable stability of the phenotype represents a novel finding for human MSCs, from any source, and supports the use of these cells as highly accessible stromal cells for both basic studies and potentially therapeutic applications such as allogeneic clinical use for musculoskeletal disorders

Dissociating tinnitus patients from healthy controls using resting-state cyclicity analysis and clustering

Chronic tinnitus is a common and sometimes debilitating condition that lacks scientific consensus on physiological models of how the condition arises as well as any known cure. In this study, we applied a novel cyclicity analysis, which studies patterns of leader-follower relationships between two signals, to resting-state functional magnetic resonance imaging (rs-fMRI) data of brain regions acquired from subjects with and without tinnitus. Using the output from the cyclicity analysis, we were able to differentiate between these two groups with 58–67% accuracy by using a partial least squares discriminant analysis. Stability testing yielded a 70% classification accuracy for identifying individual subjects’ data across sessions 1 week apart. Additional analysis revealed that the pairs of brain regions that contributed most to the dissociation between tinnitus and controls were those connected to the amygdala. In the controls, there were consistent temporal patterns across frontal, parietal, and limbic regions and amygdalar activity, whereas in tinnitus subjects, this pattern was much more variable. Our findings demonstrate a proof-of-principle for the use of cyclicity analysis of rs-fMRI data to better understand functional brain connectivity and to use it as a tool for the differentiation of patients and controls who may differ on specific traits. Chronic tinnitus is a common, yet poorly understood, condition without a known cure. Understanding differences in the functioning of brains of tinnitus patients and controls may lead to better knowledge regarding the physiology of the condition and to subsequent treatments. There are many ways to characterize relationships between neural activity in different parts of the brain. Here, we apply a novel method, called cyclicity analysis, to functional MRI data obtained from tinnitus patients and controls over a period of wakeful rest. Cyclicity analysis lends itself to interpretation as analysis of temporal orderings between elements of time-series data; it is distinct from methods like periodicity analysis or time correlation analysis in that its theoretical underpinnings are invariant to changes in time scales of the generative process. In this proof-of-concept study, we use the feature generated from the cyclicity analysis of the fMRI data to investigate group level differences between tinnitus patients and controls. Our findings indicate that temporal ordering of regional brain activation is much more consistent in the control population than in tinnitus population. We also apply methods of classification from machine learning to differentiate between the two populations with moderate amount of success

Imaging and Demography of the Host Galaxies of High-Redshift Type Ia Supernovae

We present the results of a study of the host galaxies of high redshift Type Ia supernovae (SNe Ia). We provide a catalog of 18 hosts of SNe Ia observed with the Hubble Space Telescope (HST) by the High-z Supernova Search Team (HZT), including images, scale-lengths, measurements of integrated (Hubble equivalent) BVRIZ photometry in bands where the galaxies are brighter than m ~ 25 mag, and galactocentric distances of the supernovae. We compare the residuals of SN Ia distance measurements from cosmological fits to measurable properties of the supernova host galaxies that might be expected to correlate with variable properties of the progenitor population, such as host galaxy color and position of the supernova. We find mostly null results; the current data are generally consistent with no correlations of the distance residuals with host galaxy properties in the redshift range 0.42 < z < 1.06. Although a subsample of SN hosts shows a formally significant (3-sigma) correlation between apparent V-R host color and distance residuals, the correlation is not consistent with the null results from other host colors probed by our largest samples. There is also evidence for the same correlations between SN Ia properties and host type at low redshift and high redshift. These similarities support the current practice of extrapolating properties of the nearby population to high redshifts pending more robust detections of any correlations between distance residuals from cosmological fits and host properties.Comment: 35 pages, 12 figures, 4 tables, accepted for publication in A

Exome Sequencing of a Multigenerational Human Pedigree

Over the next few years, the efficient use of next-generation sequencing (NGS) in human genetics research will depend heavily upon the effective mechanisms for the selective enrichment of genomic regions of interest. Recently, comprehensive exome capture arrays have become available for targeting approximately 33 Mb or ∼180,000 coding exons across the human genome. Selective genomic enrichment of the human exome offers an attractive option for new experimental designs aiming to quickly identify potential disease-associated genetic variants, especially in family-based studies. We have evaluated a 2.1 M feature human exome capture array on eight individuals from a three-generation family pedigree. We were able to cover up to 98% of the targeted bases at a long-read sequence read depth of ≥3, 86% at a read depth of ≥10, and over 50% of all targets were covered with ≥20 reads. We identified up to 14,284 SNPs and small indels per individual exome, with up to 1,679 of these representing putative novel polymorphisms. Applying the conservative genotype calling approach HCDiff, the average rate of detection of a variant allele based on Illumina 1 M BeadChips genotypes was 95.2% at ≥10x sequence. Further, we propose an advantageous genotype calling strategy for low covered targets that empirically determines cut-off thresholds at a given coverage depth based on existing genotype data. Application of this method was able to detect >99% of SNPs covered ≥8x. Our results offer guidance for “real-world” applications in human genetics and provide further evidence that microarray-based exome capture is an efficient and reliable method to enrich for chromosomal regions of interest in next-generation sequencing experiments