Analysis of Tyrosine Kinase Inhibitor-Mediated Decline in Contractile Force in Rat Engineered Heart Tissue

Introduction

Left ventricular dysfunction is a frequent and potentially severe side effect of many tyrosine kinase inhibitors (TKI). The mode of toxicity is not identified, but may include impairment of mitochondrial or sarcomeric function, autophagy or angiogenesis, either as an on-target or off-target mechanism.

Methods and Results

We studied concentration-response curves and time courses for nine TKIs in three-dimensional, force generating engineered heart tissue (EHT) from neonatal rat heart cells. We detected a concentration- and time-dependent decline in contractile force for gefitinib, lapatinib, sunitinib, imatinib, sorafenib, vandetanib and lestaurtinib and no decline in contractile force for erlotinib and dasatinib after 96 hours of incubation. The decline in contractile force was associated with an impairment of autophagy (LC3 Western blot) and appearance of autophagolysosomes (transmission electron microscopy).

Conclusion

This study demonstrates the feasibility to study TKI-mediated force effects in EHTs and identifies an association between a decline in contractility and inhibition of autophagic flux.     

Patterns of Song across Natural and Anthropogenic Soundscapes Suggest That White-Crowned Sparrows Minimize Acoustic Masking and Maximize Signal Content

Soundscapes pose both evolutionarily recent and long-standing sources of selection on acoustic communication. We currently know more about the impact of evolutionarily recent human-generated noise on communication than we do about how natural sounds such as pounding surf have shaped communication signals over evolutionary time. Based on signal detection theory, we hypothesized that acoustic phenotypes will vary with both anthropogenic and natural background noise levels and that similar mechanisms of cultural evolution and/or behavioral flexibility may underlie this variation. We studied song characteristics of white-crowned sparrows (Zonotrichia leucophrys nuttalli) across a noise gradient that includes both anthropogenic and natural sources of noise in San Francisco and Marin counties, California, USA. Both anthropogenic and natural soundscapes contain high amplitude low frequency noise (traffic or surf, respectively), so we predicted that birds would produce songs with higher minimum frequencies in areas with higher amplitude background noise to avoid auditory masking. We also anticipated that song minimum frequencies would be higher than the projected lower frequency limit of hearing based on site-specific masking profiles. Background noise was a strong predictor of song minimum frequency, both within a local noise gradient of three urban sites with the same song dialect and cultural evolutionary history, and across the regional noise gradient, which encompasses 11 urban and rural sites, several dialects, and several anthropogenic and natural sources of noise. Among rural sites alone, background noise tended to predict song minimum frequency, indicating that urban sites were not solely responsible for driving the regional pattern. These findings support the hypothesis that songs vary with local and regional soundscapes regardless of the source of noise. Song minimum frequency from five core study sites was also higher than the lower frequency limit of hearing at each site, further supporting the hypothesis that songs vary to transmit through noise in local soundscapes. Minimum frequencies leveled off at noisier sites, suggesting that minimum frequencies are constrained to an upper limit, possibly to retain the information content of wider bandwidths. We found evidence that site noise was a better predictor of song minimum frequency than territory noise in both anthropogenic and natural soundscapes, suggesting that cultural evolution rather than immediate behavioral flexibility is responsible for local song variation. Taken together, these results indicate that soundscapes shape song phenotype across both evolutionarily recent and long-standing soundscapes.     

The role of microaerophilic Fe‐oxidizing micro‐organisms in producing banded iron formations

Despite the historical and economic significance of banded iron formations (BIFs), we have yet to resolve the formation mechanisms. On modern Earth, neutrophilic microaerophilic Fe‐oxidizing micro‐organisms (FeOM) produce copious amounts of Fe oxyhydroxides, leading us to wonder whether similar organisms played a role in producing BIFs. To evaluate this, we review the current knowledge of modern microaerophilic FeOM in the context of BIF paleoenvironmental studies. In modern environments wherever Fe(II) and O₂ co‐exist, microaerophilic FeOM proliferate. These organisms grow in a variety of environments, including the marine water column redoxcline, which is where BIF precursor minerals likely formed. FeOM can grow across a range of O₂ concentrations, measured as low as 2 μm to date, although lower concentrations have not been tested. While some extant FeOM can tolerate high O₂ concentrations, many FeOM appear to prefer and thrive at low O₂ concentrations (~3–25 μm ). These are similar to the estimated dissolved O₂ concentrations in the few hundred million years prior to the ‘Great Oxidation Event’ (GOE). We compare biotic and abiotic Fe oxidation kinetics in the presence of varying levels of O₂ and show that microaerophilic FeOM contribute substantially to Fe oxidation, at rates fast enough to account for BIF deposition. Based on this synthesis, we propose that microaerophilic FeOM were capable of playing a significant role in depositing the largest, most well‐known BIFs associated with the GOE, as well as afterward when global O₂ levels increased.     

Fast and cloning‐free CRISPR/Cas9‐mediated genomic editing in mammalian cells

CHoP‐In (CRISPR/Cas9‐mediated Homology‐independent PCR‐product integration) is a fast, non‐homologous end‐joining based, strategy for genomic editing in mammalian cells. There is no requirement for cloning in generation of the integration donor, instead the desired integration donor is produced as a polymerase chain reaction (PCR) product, flanked by the Cas9 recognition sequences of the target locus. When co‐transfected with the cognate Cas9 and guide RNA, double strand breaks are introduced at the target genomic locus and at both ends of the PCR product. This allows incorporation into the genomic locus via hon‐homologous end joining. The approach is versatile, allowing N‐terminal, C‐terminal or internal tag integration and gives predictable genomic integrations, as demonstrated for a selection of well characterised membrane trafficking proteins. The lack of donor vectors offers advantages over existing methods in terms of both speed and hands‐on time. As such this approach will be a useful addition to the genome editing toolkit of those working in mammalian cell systems.     

Role of unusual O-glycans in intercellular signaling

In the last two decades, our knowledge of the role of glycans in development and signal transduction has expanded enormously. While most work has focused on the importance of N-linked or mucin-type O-linked glycosylation, recent work has highlighted the importance of several more unusual forms of glycosylation that are the focus of this review. In particular, the ability of O-fucose glycans on the epidermal growth factor-like (EGF) repeats of Notch to modulate signaling places glycosylation alongside phosphorylation as a means to modulate protein-protein interactions and their resultant downstream signals. The recent discovery that O-glucose modification of Notch EGF repeats is also required for Notch function has further expanded the range of glycosylation events capable of modulating Notch signaling. The prominent role of Notch during development and in later cell-fate decisions underscores the importance of these modifications in human biology. The role of glycans in intercellular signaling events is only beginning to be understood and appears ready to expand into new areas with the discovery that thrombospondin type 1 repeats are also modified with O-fucose glycans. Finally, a rare form of glycosylation called C-mannosylation modifies tryptophans in some signaling competent molecules and may be a further layer of complexity in the field. We will review each of these areas focusing on the glycan structures produced, the consequence of their presence, and the enzymes responsible.     

Draft Genome Sequences of Yersinia pestis Isolates from Natural Foci of Endemic Plague in China

To gain insights into the evolutionary origin, emergence, and pathogenicity of the etiologic agent of plague, we have sequenced the genomes of four Yersinia pestis strains isolated from the zoonotic rodent reservoir in foci of endemic plague in China. These resources enable in-depth studies of Y. pestis sequence variations and detailed whole-genome comparisons of very closely related genomes from the supposed site of the origin and the emergence of global pandemics of plague.Here we report on the genomes of Yersinia pestis strains B42003004, K1973002, E1979001, and F1991016, which represent a sample of the genetic diversity found in four foci of endemic plague in China (24). Y. pestis bv. orientalis strain F1991016 was isolated in 1991 from Cangyuan County, China, from a rat (Rattus flavipectus), and Y. pestis bv. antiqua strain E1979001 was isolated in 1979 from Jianchuan, China, from a vole (Eothenomys miletus). Both Y. pestis strains K1973002 and B42003004 of biovars medievalis and antiqua, respectively, originate from marmota species (Marmota himalayana Hetian 1973; Marmota baibacina Wenquan 2003) (24). Genome analyses of these key isolates outline the details of microevolution of the plague bacterium, as these isolates represent important evolutionary milestones of the species, which is thought to have originated in Central Asia as a clonal descendant of Yersinia pseudotuberculosis (1). Genomic DNA was subjected to whole-genome shotgun sequencing and closure strategies as previously described (15). Plasmid (pHOS2) and fosmid (pCC1fos) libraries were constructed, with insert sizes of 4 to 6 kb and 30 to 40 kb, respectively. An average of 67,000 high-quality Sanger reads (total, 268,160) was obtained with an 860-bp average read length. The genomes with an average 12-fold read coverage depth were assembled using a Celera Assembler (11) and manually annotated using Manatee (http://manatee.sourceforge.net/). Genomic architectures were compared using Mauve (5, 18), and proteomes were analyzed with the BLAST score ratio tool (17).The young evolutionary history of the species and resulting homogenous population structure is reflected in a high degree of proteome conservation between the sequenced isolates and the modern strain CO92 (16). Y. pestis pathogenicity is anchored in its mobile inventory, and typically, isolates harbor three virulence plasmids, the species-specific plasminogen activator and murine toxin plasmids and the low-calcium-response plasmid pCD (23). Their pCD-borne lcrV antigen shows a genetic makeup identical to that of CO92 (2, 16). The insertion sequence element expansion clearly distinguishes these Central Asian isolates from the progenitor Y. pseudotuberculosis (3, 8). Comprehensive analyses reveal a lack of genome-wide synteny and suggest massive intrachromosomal rearrangements, a characteristic feature of Y. pestis genome evolution (6, 8). Besides insertion sequence element abundance, we observed isolate-specific propagation patterns that not only shaped the reorganization of the genomic architecture but also are known to drive microevolutionary adaptation in Y. pestis (4, 9, 14, 21, 24). Based upon the phenotypic and genotypic features that differentiate these isolates (13, 20, 24), B42003004 belongs to the most ancient Y. pestis lineage known to exist in China; hence, it is phylogenetically thought to be closest to the species progenitor Y. pseudotuberculosis (22). We studied metabolic genes that determine their biovar classification and investigated the underlying genetic determinants (24). Isolate K1973002 is defective in the nitrate reductase napA gene, similar to strain KIM (7), and represents the results of the evolutionary processes implicated in the biovar conversion from antiqua to medievalis. Isolate F1991016 carries an in-frame deletion in the glycerol-3-phosphate dehydrogenase glpD gene (19), similar to strain CO92 (16), and characteristic of the antiqua-to-orientalis conversion. The observed genetic traits strengthen the hypothesis that biovars medievalis and orientalis arose through parallel evolution from a glycerol- and nitrate-positive antiqua progenitor due to the acquisition of independent mutations (1, 10, 14). Variable-number tandem-nucleotide-repeat alleles (12) (allele K, K1973002; allele K, B42003004; allele P, E1979001; allele G, F1991016) are not biovar specific and are not discriminative enough to differentiate these isolates, which clearly supports a population-based phylogeny, as introduced by Achtman et al. (1).The whole-genome draft sequences of these evolutionary key isolates of Y. pestis will facilitate additional bioinformatic and phylogenetic analyses. The availability of high-quality Sanger sequences is crucial to resolve the genetically homogenous population structure and to shed light on Y. pestis speciation. Understanding the plasticity and genome dynamics further aids in forensic and epidemiological analyses by setting up the basis for an accurate and robust typing system for plague surveillance and promotes diagnostics development and control measures.     

Structural and Mechanistic Insights into Lunatic Fringe from a Kinetic Analysis of Enzyme Mutants

The Notch receptor is critical for proper development where it orchestrates numerous cell fate decisions. The Fringe family of β1,3-N-acetylglucosaminyltransferases are regulators of this pathway. Fringe enzymes add N-acetylglucosamine to O-linked fucose on the epidermal growth factor repeats of Notch. Here we have analyzed the reaction catalyzed by Lunatic Fringe (Lfng) in detail. A mutagenesis strategy for Lfng was guided by a multiple sequence alignment of Fringe proteins and solutions from docking an epidermal growth factor-like O-fucose acceptor substrate onto a homology model of Lfng. We targeted three main areas as follows: residues that could help resolve where the fucose binds, residues in two conserved loops not observed in the published structure of Manic Fringe, and residues predicted to be involved in UDP-N-acetylglucosamine (UDP-GlcNAc) donor specificity. We utilized a kinetic analysis of mutant enzyme activity toward the small molecule acceptor substrate 4-nitrophenyl-α-l-fucopyranoside to judge their effect on Lfng activity. Our results support the positioning of O-fucose in a specific orientation to the catalytic residue. We also found evidence that one loop closes off the active site coincident with, or subsequent to, substrate binding. We propose a mechanism whereby the ordering of this short loop may alter the conformation of the catalytic aspartate. Finally, we identify several residues near the UDP-GlcNAc-binding site, which are specifically permissive toward UDP-GlcNAc utilization.Defects in Notch signaling have been implicated in numerous human diseases, including multiple sclerosis (1), several forms of cancer (2-4), cerebral autosomal dominant arteriopathy with sub-cortical infarcts and leukoencephalopathy (5), and spondylocostal dysostosis (SCD)³ (6-8). The transmembrane Notch signaling receptor is activated by members of the DSL (Delta, Serrate, Lag2) family of ligands (9, 10). In the endoplasmic reticulum, O-linked fucose glycans are added to the epidermal growth factor-like (EGF) repeats of the Notch extracellular domain by protein O-fucosyltransferase 1 (11-13). These O-fucose monosaccharides can be elongated in the Golgi apparatus by three highly conserved β1,3-N-acetylglucosaminyltransferases of the Fringe family (Lunatic (Lfng), Manic (Mfng), and Radical Fringe (Rfng) in mammals) (14-16). The formation of this GlcNAc-β1,3-Fuc-α1, O-serine/threonine disaccharide is necessary and sufficient for subsequent elongation to a tetrasaccharide (15, 19), although elongation past the disaccharide in Drosophila is not yet clear (20, 21). Elongation of O-fucose by Fringe is known to potentiate Notch signaling from Delta ligands and inhibit signaling from Serrate ligands (22). Delta ligands are termed Delta-like (Delta-like1, -2, and -4) in mammals, and the homologs of Serrate are known as Jagged (Jagged1 and -2) in mammals. The effects of Fringe on Drosophila Notch can be recapitulated in Notch ligand in vitro binding assays using purified components, suggesting that the elongation of O-fucose by Fringe alters the binding of Notch to its ligands (21). Although Fringe also appears to alter Notch-ligand interactions in mammals, the effects of elongation of the glycan past the O-fucose monosaccharide is more complicated and appears to be cell type-, receptor-, and ligand-dependent (for a recent review see Ref. 23).The Fringe enzymes catalyze the transfer of GlcNAc from the donor substrate UDP-α-GlcNAc to the acceptor fucose, forming the GlcNAc-β1,3-Fuc disaccharide (14-16). They belong to the GT-A-fold of inverting glycosyltransferases, which includes N-acetylglucosaminyltransferase I and β1,4-galactosyltransferase I (17, 18). The mechanism is presumed to proceed through the abstraction of a proton from the acceptor substrate by a catalytic base (Asp or Glu) in the active site. This creates a nucleophile that attacks the anomeric carbon of the nucleotide-sugar donor, inverting its configuration from α (on the nucleotide sugar) to β (in the product) (24, 25). The enzyme then releases the acceptor substrate modified with a disaccharide and UDP. The Mfng structure (26) leaves little doubt as to the identity of the catalytic residue, which in all likelihood is aspartate 289 in mouse Lfng (we will use numbering for mouse Lunatic Fringe throughout, unless otherwise stated). The structure of Mfng with UDP-GlcNAc soaked into the crystals (26) showed density only for the UDP portion of the nucleotide-sugar donor and no density for two loops flanking either side of the active site. The presence of flexible loops that become ordered upon substrate binding is a common observation with glycosyltransferases in the GT-A fold family (18, 25). Density for the entire donor was observed in the structure of rabbit N-acetylglucosaminyltransferase I (27). In this case, ordering of a previously disordered loop upon UDP-GlcNAc binding may have contributed to increased stability of the donor. In the case of bovine β1,4-galactosyltransferase I, a section of flexible random coil from the apo-structure was observed to change its conformation to α-helical upon donor substrate binding (28). Both loops in Lfng are highly conserved, and we have mutated a number of residues in each to test the hypothesis that they interact with the substrates. The mutagenesis strategy was also guided by docking of an EGF-O-fucose acceptor substrate into the active site of the Lfng model as well as comparison of the Lfng model with a homology model of the β1,3-glucosyltransferase (β3GlcT) that modifies O-fucose on thrombospondin type 1 repeats (29, 30). The β3GlcT is predicted to be a GT-A fold enzyme related to the Fringe family (17, 18, 29).     

Transrectal Doppler sonography for evaluation of uterine blood flow throughout pregnancy in 13 cows

Once weekly from 30 to 270 days of gestation in 13 cows, Doppler ultrasound scanning (triplex Doppler system) was done to assess blood-flow parameters of both median uterine arteries. Resistance, velocity and volume indices were measured. Resistance index values were negatively correlated to all other blood-flow parameters (P<0.05), but there were positive correlations between velocity and volume indices (P<0.05). Resistance indices were lower, and velocity and volume indices were significantly higher in the median uterine artery ipsilateral versus contralateral to the fetus. Resistance indices decreased continuously during the first 36 weeks of pregnancy. Velocity values rose three-fold, whereas the area increased 20-fold and the volume increased 17-fold by the end of gestation (P<0.05). Birth weight of calves was positively correlated with blood-flow volume (r=0.34) but negatively correlated with the resistance index (r=-0.45). There were no significant differences between male versus female calves (at any stage of gestation) in the resistance, time-average maximum velocity, and volume indices (P>0.05). In conclusion, arterial blood flow was monitored with transrectal Doppler sonography in both median uterine arteries weekly throughout pregnancy in cattle; this could be very valuable for monitoring pregnancies at high risk for abnormalities of the placenta, fetus or both, e.g. cloned calves.