Larger Genomes Linked to Slower Development and Loss of Late-Developing Traits

Genome size varies widely among organisms and is known to affect vertebrate development, morphology, and physiology. In amphibians, genome size is hypothesized to contribute to loss of late-forming structures, although this hypothesis has mainly been discussed in salamanders. Here we estimated genome size for 22 anuran species and combined this novel dataset with existing genome size data for an additional 234 anuran species to determine whether larger genome size is associated with loss of a late-forming anuran sensory structure, the tympanic middle ear. We established that genome size is negatively correlated with development rate across 90 anuran species and found that genome size evolution is correlated with evolutionary loss of the middle ear bone (columella) among 241 species (224 eared and 17 earless). We further tested whether the development of the tympanic middle ear could be constrained by large cell sizes and small body sizes during key stages of tympanic middle ear development (metamorphosis). Together, our evidence suggests that larger genomes, slower development rate, and smaller body sizes at metamorphosis may contribute to the loss of the anuran tympanic middle ear. We conclude that increases in anuran genome size, although less drastic than in salamanders, may affect development of late-forming traits

Neural Stem Cells as a Novel Platform for Tumor-Specific Delivery of Therapeutic Antibodies

Recombinant monoclonal antibodies have emerged as important tools for cancer therapy. Despite the promise shown by antibody-based therapies, the large molecular size of antibodies limits their ability to efficiently penetrate solid tumors and precludes efficient crossing of the blood-brain-barrier into the central nervous system (CNS). Consequently, poorly vascularized solid tumors and CNS metastases cannot be effectively treated by intravenously-injected antibodies. The inherent tumor-tropic properties of human neural stem cells (NSCs) can potentially be harnessed to overcome these obstacles and significantly improve cancer immunotherapy. Intravenously-delivered NSCs preferentially migrate to primary and metastatic tumor sites within and outside the CNS. Therefore, we hypothesized that NSCs could serve as an ideal cellular delivery platform for targeting antibodies to malignant tumors., and can deliver antibodies to human breast cancer xenografts in mice.Taken together, these results suggest that NSCs modified to secrete HER2-targeting antibodies constitute a promising novel platform for targeted cancer immunotherapy. Specifically, this NSC-mediated antibody delivery system has the potential to significantly improve clinical outcome for patients with HER2-overexpressing breast cancer

Womack_etal_2019_GenomeSizeDataTable_ForDryad

Data used and relevant references for Womack et al. 2019 publication in American Naturalist

The role of the C2A domain of synaptotagmin 1 in asynchronous neurotransmitter release.

Following nerve stimulation, there are two distinct phases of Ca2+-dependent neurotransmitter release: a fast, synchronous release phase, and a prolonged, asynchronous release phase. Each of these phases is tightly regulated and mediated by distinct mechanisms. Synaptotagmin 1 is the major Ca2+ sensor that triggers fast, synchronous neurotransmitter release upon Ca2+ binding by its C2A and C2B domains. It has also been implicated in the inhibition of asynchronous neurotransmitter release, as blocking Ca2+ binding by the C2A domain of synaptotagmin 1 results in increased asynchronous release. However, the mutation used to block Ca2+ binding in the previous experiments (aspartate to asparagine mutations, sytD-N) had the unintended side effect of mimicking Ca2+ binding, raising the possibility that the increase in asynchronous release was directly caused by ostensibly constitutive Ca2+ binding. Thus, rather than modulating an asynchronous sensor, sytD-N may be mimicking one. To directly test the C2A inhibition hypothesis, we utilized an alternate C2A mutation that we designed to block Ca2+ binding without mimicking it (an aspartate to glutamate mutation, sytD-E). Analysis of both the original sytD-N mutation and our alternate sytD-E mutation at the Drosophila neuromuscular junction showed differential effects on asynchronous release, as well as on synchronous release and the frequency of spontaneous release. Importantly, we found that asynchronous release is not increased in the sytD-E mutant. Thus, our work provides new mechanistic insight into synaptotagmin 1 function during Ca2+-evoked synaptic transmission and demonstrates that Ca2+ binding by the C2A domain of synaptotagmin 1 does not inhibit asynchronous neurotransmitter release in vivo

NSCs target breast carcinoma and can deliver anti-HER2 antibody <i>in vivo</i>.

<p>Confocal fluorescence micrographs of tumor sections from MCF7/HER2 xenografts. First three panels in the upper row show the presence of CM-DiI-labeled red NSCs (HB1.F3, HB1.F3.Ad-H2IgG, HB1.F3.Lenti-H2IgG, respectively) in tumors 4 days after intravenous injection. The fourth panel of the upper row shows tumor with no red NSCs in mice treated with trastuzumab alone (sporadic small red dots not associated with cells are visible as autofluorescence background). Middle row shows tumor sections stained with FITC-conjugated anti-human IgG (green). Bar, 50 µm. Insets are 2× magnification (Bar, 20 µm). Bottom row shows confirmation of the presence or absence of HB1.F3 NSCs within tumors by PCR detection of a DNA amplicon (293 bp) of the v<i>-myc</i> transgene, a unique identifier of the HB1.F3 cell line.</p

NSC-secreted anti-HER2 antibody is functionally equivalent to trastuzumab.

<p>Flow cytometric analysis (<b>A</b>) of MCF7, MCF7/HER2, and BT474 cells labeled with F3-IgG, trastuzumab, or a human IgG isotype control antibody. Graphs show mean fluorescence intensity (MFI) of labeled cells. Inhibition of cell proliferation (<b>B</b>) of MCF7, MCF7/HER2, or BT474 cells treated for 6 days with F3-IgG, trastuzumab, or isotype control antibody. Graphs show proliferation as a percentage of untreated cells.</p

NSC-secreted human IgG specifically binds HER2.

<p>Transfected NSCs were co-cultured with CM-DiI-labeled (red) MCF7 (<b>A</b>) or MCF7/HER2 cells (<b>B</b>) and stained with FITC-conjugated anti-human IgG (green) and DAPI (blue). Arrows indicate NSCs expressing human IgG that does not bind to adjacent MCF7 control cells (<b>A</b>) or areas of NSC-secreted human IgG bound to MCF7/HER2 target cells (<b>B</b>). Bar, 50 µm. Flow cytometric analysis of BT474, MCF7/HER2, or MCF7 target cells after incubation with supernatant from HB1.F3.H2IgG, HB1.F3.Adeno-H2IgG, or HB1.F3.Lenti-H2IgG (<b>C</b>), followed by incubation with FITC-conjugated anti-human IgG (blue histograms). Red histograms on each graph show target cells incubated with supernatant from unmodified HB1.F3 NSCs as a negative control.</p

<i>In vitro</i> migration of NSCs to breast carcinoma conditioned media.

<p>Migration of parental NSCs and anti-HER2-transfected HB1.F3 NSCs to breast tumor-conditioned media in an <i>in vitro</i> chemotaxis assay. In this assay, bovine serum albumin (BSA) was used as a negative control for chemotaxis. Both parental and transfected NSC lines preferentially migrated to MCF7/HER2 compared to negative control (2% BSA) (<i>p</i><0.01).</p