Characterization of the mode of action of eCry1Gb.1Ig, a fall armyworm (Spodoptera frugiperda) active protein, with a novel site of action.

Insect pests cause immense agronomic losses worldwide. One of the most destructive of major crops is the Fall Armyworm (Spodoptera frugiperda, FAW). The ability to migrate long distances, a prodigious appetite, and a demonstrated ability to develop resistance to insecticides, make it a difficult target to control. Insecticidal proteins, for example those produced by the bacterium Bacillus thuringiensis, are among the safest and most effective insect control agents. Genetically modified (GM) crops expressing such proteins are a key part of a successful integrated pest management (IPM) program for FAW. However, due to the development of populations resistant to commercialized GM products, new GM traits are desperately needed. Herein, we describe a further characterization of the newly engineered trait protein eCry1Gb.1Ig. Similar to other well characterized Cry proteins, eCry1Gb.1Ig is shown to bind FAW midgut cells and induce cell-death. Binding competition assays using trait proteins from other FAW-active events show a lack of competition when binding FAW brush border membrane vesicles (BBMVs) and when utilizing non-pore-forming versions as competitors in in vivo bioassays. Similarly, insect cell lines expressing SfABCC2 and SfABCC3 (well characterized receptors of existing commercial Cry proteins) are insensitive to eCry1Gb.1Ig. These findings are consistent with results from our previous work showing that eCry1Gb.1Ig is effective in controlling insects with resistance to existing traits. This underscores the value of eCry1Gb.1Ig as a new GM trait protein with a unique site-of-action and its potential positive impact to global food production.

Observations of ocean spice and isopycnal tilt sound-speed structures in the mixed layer and upper ocean and their impacts on acoustic propagation.

A 400-m deep and 970 km long conductivity, temperature, depth section in the Northeast Pacific Ocean is decomposed into sound-speed variations associated with tilting isopycnals and ocean spice. The vertical distribution of sound-speed variance from these two processes shows significant fluctuations in the mixed layer (ML) and transition layer (TRL) below. Acoustic simulations at 400 and 1000 Hz are conducted with the decomposed fields to quantify their relative impact on upper ocean propagation for source locations in the ML and TRL. The low frequency simulations show that localized scattering processes dominate the propagation while higher frequencies experience more diffuse scattering. For propagation in the ML, spice generates the most loss while tilt can reduce loss when combined with spice. Statistics further show that energy can couple into and out of the ML duct depending on source depth and frequency.

Observations of the space/time scales of Beaufort sea acoustic duct variability and their impact on transmission loss via the mode interaction parameter.

The Beaufort duct (BD) is a subsurface sound channel in the western Arctic Ocean formed by cold Pacific Winter Water (PWW) sandwiched between warmer Pacific Summer Water (PSW) and Atlantic Water (AW). Sound waves can be trapped in this duct and travel long distances without experiencing lossy surface/ice interactions. This study analyzes BD vertical and temporal variability using moored oceanographic measurements from two yearlong acoustic transmission experiments (2016-2017 and 2019-2020). The focus is on BD normal mode propagation through observed ocean features, such as eddies and spicy intrusions, where direct numerical simulations and the mode interaction parameter (MIP) are used to quantify ducted mode coupling strength. The observations show strong PSW sound speed variability, weak variability in the PWW, and moderate variability in the AW, with typical time scales from days to weeks. For several hundreds Hertz propagation, the BD modes are relatively stable, except for rare episodes of strong sound speed perturbations. The MIP identifies a resonance condition such that the likelihood of coupling is greatest when there is significant sound speed variability in the horizontal wave number band 1/11

Direct capture of neutralized RBD enables rapid point-of-care assessment of SARS-CoV-2 neutralizing antibody titer.

Neutralizing antibody (NAb) titer is a key biomarker of protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but point-of-care methods for assessing NAb titer are not widely available. Here, we present a lateral flow assay that captures SARS-CoV-2 receptor-binding domain (RBD) that has been neutralized from binding angiotensin-converting enzyme 2 (ACE2). Quantification of neutralized RBD in this assay correlates with NAb titer from vaccinated and convalescent patients. This methodology demonstrated superior performance in assessing NAb titer compared with either measurement of total anti-spike immunoglobulin G titer or quantification of the absolute reduction in binding between ACE2 and RBD. Our testing platform has the potential for mass deployment to aid in determining at population scale the degree of protective immunity individuals may have following SARS-CoV-2 vaccination or infection and can enable simple at-home assessment of NAb titer.

Beaufort Sea observations of 11 to 12.5 kHz surface pulse reflections near 50 degree grazing angle from 2016 to 2017.

Sea-surface acoustic scattering is investigated using observations from the 2016-2017 Canada Basin Acoustic Propagation Experiment. The motions of the low-frequency acoustic source and/or receiver moorings were measured using long-baseline acoustic navigation systems in which the signals transmitted once per hour by the mooring instruments triggered high-frequency replies from the bottom-mounted transponders. The moorings recorded these replies, giving the direct path and single-bounce surface-reflected arrivals, which have grazing angles near 50°. The reflected signals are used here to quantify the surface scattering statistics in an opportunistic effort to infer the changing ice characteristics as a function of time and space. Five scattering epochs are identified: (1) open water, (2) initial ice formation, (3) ice solidification, (4) ice thickening, and (5) ice melting. Significant changes in the ice scattering observables are seen using the arrival angle, moment of reflected intensity and its probability density function, and pulse time spread. The largest changes took place during the formation, solidification, and melting. The statistical characteristics across the experimental region are similar, suggesting consistent ice properties. To place the results in some physical context, they are interpreted qualitatively using notions of the partial and fully saturated wave fields, a Kirchhoff-like approximation for the rough surface, and a thin elastic layer reflection coefficient model.

Observations of scatter from surface reflectors with Doppler sensitive probe signals.

Previous analyses of surface scatter from the at-sea KAM11 experiment were made with linear frequency modulated waveforms that emphasized a single slope direction for arrivals in time-varying impulse response estimates. This analysis of Doppler sensitive waveform transmissions, made in the same geometry, resolves additional scatter arrivals with opposite slope. The different Doppler shifts in scatter observations are related to dispersed, naturally occurring, sea surface features that reflect the transmitted waveform to the receiver. The positions of these surface reflectors are estimated from the delay and Doppler shift of the observed arrivals without needing a receiving array with high spatial resolution.

BlobToolKit - Interactive Quality Assessment of Genome Assemblies.

Reconstruction of target genomes from sequence data produced by instruments that are agnostic as to the species-of-origin may be confounded by contaminant DNA. Whether introduced during sample processing or through co-extraction alongside the target DNA, if insufficient care is taken during the assembly process, the final assembled genome may be a mixture of data from several species. Such assemblies can confound sequence-based biological inference and, when deposited in public databases, may be included in downstream analyses by users unaware of underlying problems. We present BlobToolKit, a software suite to aid researchers in identifying and isolating non-target data in draft and publicly available genome assemblies. BlobToolKit can be used to process assembly, read and analysis files for fully reproducible interactive exploration in the browser-based Viewer. BlobToolKit can be used during assembly to filter non-target DNA, helping researchers produce assemblies with high biological credibility. We have been running an automated BlobToolKit pipeline on eukaryotic assemblies publicly available in the International Nucleotide Sequence Data Collaboration and are making the results available through a public instance of the Viewer at https://blobtoolkit.genomehubs.org/view We aim to complete analysis of all publicly available genomes and then maintain currency with the flow of new genomes. We have worked to embed these views into the presentation of genome assemblies at the European Nucleotide Archive, providing an indication of assembly quality alongside the public record with links out to allow full exploration in the Viewer.

The European Nucleotide Archive in 2019.

The European Nucleotide Archive (ENA, https://www.ebi.ac.uk/ena) at the European Molecular Biology Laboratory's European Bioinformatics Institute provides open and freely available data deposition and access services across the spectrum of nucleotide sequence data types. Making the world's public sequencing datasets available to the scientific community, the ENA represents a globally comprehensive nucleotide sequence resource. Here, we outline ENA services and content in 2019 and provide an insight into selected key areas of development in this period.

The European Nucleotide Archive in 2018.

The European Nucleotide Archive (ENA; https://www.ebi.ac.uk/ena), provided from EMBL-EBI, has for more than three decades been responsible for archiving the world's public sequencing data and presenting this important resource to the scientific community to support and accelerate the global research effort. Here, we outline ENA services and content in 2018 and provide an overview of a selection of focus areas of development work: extending data coordination services around ENA, sequence submissions through template expansion, early pre-submission validation tools and our move towards a new browser and retrieval infrastructure.

Calibration of vertical array tilt using snapping shrimp sound.

Snapping shrimp are the dominant biological source of high-frequency (>2 kHz) ambient noise in warm coastal waters. In a recent experiment, the highly impulsive signals produced by shrimp snaps were recorded continually by a large-aperture vertical array (56 m) that was bottom-moored in 100-m deep shallow water. Assuming the array vertical, initial localization of individual snaps based on wavefront curvature along the array indicated that all snaps came from either above or beneath the flat seabed. By constraining all snaps to originate from the seabed, several hundred snaps within a radius of 500 m from the array over a 20-s window were detected successfully and localized in the three-dimensional space of time-of-arrival, range, and array tilt. Since the estimated array tilt for each snap is a projection of the absolute array tilt onto the nominal array-snap plane, the maximal tilt in the range and tilt domain corresponds to the absolute array tilt. Both simulations and data demonstrate that snapping shrimp can be exploited as a source of opportunity for calibration of vertical array tilt.

Acoustic scattering comparison of Kirchhoff approximation to Rayleigh-Fourier method for sinusoidal surface waves at low grazing angles.

The Fourier series method for implementing the Rayleigh hypothesis [Rayleigh-Fourier method (RFM)] is used as a reference solution to assess the Kirchhoff approximation of the Helmholtz integral [Helmholtz-Kirchhoff approximation (HKA)] for modeling broadband scatter from sinusoidal surfaces at low grazing angles. The HKA is a valuable solution because it has an eigen-ray interpretation without unbounded caustic amplitudes and discontinuous shadow zones. Plane wave studies of the HKA, however, show it becomes inaccurate at low grazing angles. This study quantifies how this limitation manifests with increasing transmission distance for time domain scattering simulations. Scattering results are compared over a complete surface wave cycle with parameters modeling sea surface-swell. The HKA agrees reasonably well with the RFM in point source calculations for limited extensions of transmission distances beyond where plane wave comparisons begin to diverge. Past these distances, HKA solutions begin to show significant over-prediction of the acoustic amplitude around late arrivals. This over-prediction is frequency dependent and eigen-ray interference offers an explanation of this behavior. Further extending the transmission range leads to a significant HKA error, and a range is found at which flat surface reflections have less error.

Human induced pluripotent stem cell-derived glial cells and neural progenitors display divergent responses to Zika and dengue infections.

Maternal Zika virus (ZIKV) infection during pregnancy is recognized as the cause of an epidemic of microcephaly and other neurological anomalies in human fetuses. It remains unclear how ZIKV accesses the highly vulnerable population of neural progenitors of the fetal central nervous system (CNS), and which cell types of the CNS may be viral reservoirs. In contrast, the related dengue virus (DENV) does not elicit teratogenicity. To model viral interaction with cells of the fetal CNS in vitro, we investigated the tropism of ZIKV and DENV for different induced pluripotent stem cell-derived human cells, with a particular focus on microglia-like cells. We show that ZIKV infected isogenic neural progenitors, astrocytes, and microglia-like cells (pMGLs), but was only cytotoxic to neural progenitors. Infected glial cells propagated ZIKV and maintained ZIKV load over time, leading to viral spread to susceptible cells. DENV triggered stronger immune responses and could be cleared by neural and glial cells more efficiently. pMGLs, when cocultured with neural spheroids, invaded the tissue and, when infected with ZIKV, initiated neural infection. Since microglia derive from primitive macrophages originating in proximity to the maternal vasculature, they may act as a viral reservoir for ZIKV and establish infection of the fetal brain. Infection of immature neural stem cells by invading microglia may occur in the early stages of pregnancy, before angiogenesis in the brain rudiments. Our data are also consistent with ZIKV and DENV affecting the integrity of the blood-brain barrier, thus allowing infection of the brain later in life.

JNK Pathway Activation Modulates Acquired Resistance to EGFR/HER2-Targeted Therapies.

Resistance limits the effectiveness of receptor tyrosine kinase (RTK)-targeted therapies. Combination therapies targeting resistance mechanisms can considerably improve response, but will require an improved understanding of when particular combinations will be effective. One common form of resistance is bypass signaling, wherein RTKs not targeted by an inhibitor can direct reactivation of pathways essential for survival. Although this mechanism of resistance is well appreciated, it is unclear which downstream signaling events are responsible. Here, we apply a combined experimental- and statistical modeling-based approach to identify a set of pathway reactivation essential for RTK-mediated bypass resistance. Differences in the downstream pathway activation provided by particular RTKs lead to qualitative differences in the capacity of each receptor to drive therapeutic resistance. We identify and validate that the JNK pathway is activated during and strongly modulates bypass resistance. These results identify effective therapeutic combinations that block bypass-mediated resistance and provide a basic understanding of this network-level change in kinase dependence that will inform the design of prognostic assays for identifying effective therapeutic combinations in individual patients. Cancer Res; 76(18); 5219-28. ©2016 AACR.

A functional variant in HOXA11-AS, a novel long non-coding RNA, inhibits the oncogenic phenotype of epithelial ovarian cancer.

The homeobox A (HOXA) region of protein-coding genes impacts female reproductive system embryogenesis and ovarian carcinogenesis. The 5-prime end of HOXA includes three long non-coding RNAs (lncRNAs) (HOXA10-AS, HOXA11-AS, and HOTTIP) that are underexplored in epithelial ovarian cancer (EOC). We evaluated whether common genetic variants in these lncRNAs are associated with EOC risk and/or have functional roles in EOC development. Using genome-wide association study data from 1,201 serous EOC cases and 2,009 controls, an exonic variant within HOXA11-AS, rs17427875 (A>T), was marginally associated with reduced serous EOC risk (OR = 0.88 (95% CI: 0.78-1.01, p = 0.06). Functional studies of ectopic expression of HOXA11-AS minor allele T in EOC cells showed decreased survival, proliferation, migration, and invasion compared to common allele A expression. Additionally, stable expression of HOXA11-AS minor allele T reduced primary tumor growth in mouse xenograft models to a greater extent than common allele A. Furthermore, HOXA11-AS expression levels were significantly lower in human EOC tumors than normal ovarian tissues (p < 0.05), suggesting that HOXA11-AS has a tumor suppressor function in EOC which may be enhanced by the T allele. These findings demonstrate for the first time a role for HOXA11-AS in EOC with effects that could be modified by germline variants.

Long non-coding RNAs (LncRNA) regulated by transforming growth factor (TGF) ß: LncRNA-hit-mediated TGFß-induced epithelial to mesenchymal transition in mammary epithelia.

Long noncoding RNAs (lncRNAs) are emerging as key regulators in various biological processes. Epithelial-to-mesenchymal transition (EMT) is a developmental process hijacked by tumor cells to depart from the primary tumor site, invade surrounding tissue, and establish distant metastases. Transforming growth factor ß (TGFß) signaling has been shown to be a major inducer of EMT and to facilitate breast cancer metastasis. However, the role of lncRNAs in this process remains largely unknown. Here we report a genome-wide lncRNA profile in mouse mammary epithelial NMuMG cells upon TGFß induction of EMT. Among 10,802 lncRNAs profiled, over 600 were up-regulated and down-regulated during the EMT, respectively. Furthermore, we identify that lncRNA-HIT (HOXA transcript induced by TGFß) mediates TGFß function, i.e. depletion of lncRNA-HIT inhibits TGFß-induced migration, invasion, and EMT in NMuMG. LncRNA-HIT is also significantly elevated in the highly metastatic 4T1 cells. Knockdown of lncRNA-HIT in 4T1 results in decrease of cell migration, invasion, tumor growth, and metastasis. E-cadherin was identified as a major target of lncRNA-HIT. Moreover, lncRNA-HIT is conserved in humans and elevated expression associates with more invasive human primary breast carcinoma. Collectively, these data suggest that a subset of lncRNAs such as lncRNA-HIT play a significant role in regulation of EMT and breast cancer invasion and metastasis, and could be potential therapeutic targets in breast cancers.

Evaluation of Tyro3 expression, Gas6-mediated Akt phosphorylation, and the impact of anti-Tyro3 antibodies in melanoma cell lines.

Tyro3, a member of the Tyro3/Axl/Mer (TAM) family of receptor tyrosine kinases, has emerged as a potential oncogene in melanoma. Here, we confirm that Tyro3 is specifically overexpressed in primary melanoma samples and show that Tyro3 is expressed at varying levels in numerous melanoma cell lines. Short hairpin RNA-mediated knockdown of Tyro3 led to significant cell death via apoptotic mechanisms in nearly all melanoma cell lines tested, regardless of the BRAF or NRAS mutation status or co-expression of Axl and/or Mer. We generated soluble and monomeric versions of the human Tyro3 extracellular domain and human Gas6 for affinity measurements and correlated these values with the level of Gas6 required to induce Tyro3 signaling in cellular assays. Calcium was critical for the correct folding of Gas6 and its binding to Tyro3. In melanoma cell lines, Gas6 induced Tyro3 phosphorylation and downstream Akt phosphorylation without apparent effects on Erk. We generated monoclonal antibodies (mAbs) against Tyro3 to examine their effect on survival signaling in melanoma cell lines. The mAbs generated against Tyro3 included nonligand blockers, partial blockers, and competitive ligand blockers. A number of weak and partial ligand blockers (all recognizing the Tyro3 Ig domains) were the most effective at blocking ligand-mediated downstream signaling of Tyro3. Overall, these data indicate that Tyro3 may confer increased survival signals in melanoma cells and can be stymied using inhibitory mAbs. These mAbs may be useful for further investigations of the role of Tyro3 in melanoma.