GE11-antigen-loaded hepatitis B virus core antigen virus-like particles efficiently bind to TNBC tumor

PurposeThis study aimed to explore the possibility of utilizing hepatitis B core protein (HBc) virus-like particles (VLPs) encapsulate doxorubicin (Dox) to reduce the adverse effect caused by its off-target and toxic side effect.MethodsHere, a triple-negative breast cancer (TNBC) tumor-targeting GE11-HBc VLP was constructed through genetic engineering. The GE11 peptide, a 12-amino-acid peptide targeting epidermal growth factor receptor (EGFR), was inserted into the surface protein loops of VLPs. The Dox was loaded into HBc VLPs by a thermal-triggered encapsulation strategy. The in vitro release, cytotoxicity, and cellular uptake of TNBC tumor-targeting GE11-HBc VLPs was then evaluated.ResultsThese VLPs possessed excellent stability, DOX loading efficiency, and preferentially released drug payload at high GSH levels. The insertion of GE11 targeting peptide caused improved cellular uptake and enhanced cell viability inhibitory in EGFR high-expressed TNBC cells.ConclusionTogether, these results highlight DOX-loaded, EGFR-targeted VLPs as a potentially useful therapeutic choice for EGFR-overexpressing TNBC

Evolution of Yin and Yang isoforms of a chromatin remodeling subunit precedes the creation of two genes

Genes can encode multiple isoforms, broadening their functions and providing a molecular substrate to evolve phenotypic diversity. Evolution of isoform function is a potential route to adapt to new environments. Here we show that de novo, beneficial alleles in the nurf-1 gene became fixed in two laboratory lineages of C. elegans after isolation from the wild in 1951, before methods of cryopreservation were developed. nurf-1 encodes an ortholog of BPTF, a large (>300 kD) multidomain subunit of the NURF chromatin remodeling complex. Using CRISPR-Cas9 genome editing and transgenic rescue, we demonstrate that in C. elegans, nurf-1 has split into two, largely non-overlapping isoforms (NURF-1.D and NURF-1.B, which we call Yin and Yang, respectively) that share only two of 26 exons. Both isoforms are essential for normal gametogenesis but have opposite effects on male/female gamete differentiation. Reproduction in hermaphrodites, which involves production of both sperm and oocytes, requires a balance of these opposing Yin and Yang isoforms. Transgenic rescue and genetic position of the fixed mutations suggest that different isoforms are modified in each laboratory strain. In a related clade of Caenorhabditis nematodes, the shared exons have duplicated, resulting in the split of the Yin and Yang isoforms into separate genes, each containing approximately 200 amino acids of duplicated sequence that has undergone accelerated protein evolution following the duplication. Associated with this duplication event is the loss of two additional nurf-1 transcripts, including the long-form transcript and a newly identified, highly expressed transcript encoded by the duplicated exons. We propose these lost transcripts are non-functional side products necessary to transcribe the Yin and Yang transcripts in the same cells. Our work demonstrates how gene sharing, through the production of multiple isoforms, can precede the creation of new, independent genes.National Institute of General Medical Sciences R01GM114170 Patrick T McGrat National Institute of General Medical Sciences R01GM121688 Ronald E Ellis.The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.S

QUANTITATIVE METHODS TO UNDERSTAND REPRODUCTIVE ISOLATION THAT CONTRIBUTE TO SPECIATION

Ever since Darwin’s qualitative theory of the origin of species, there is growing demand for quantitative methods to study mechanisms underlying the speciation process. One key component towards new species formation is reproductive isolation. Reproductive isolation can be either pre-mating (e.g. mating behavior difference) or post-mating (e.g. Dobzhansky-Muller sites). In this thesis, I will present novel quantitative methods developed to study two aspects of reproductive isolation: mating rituals difference in Lake Malawi cichlids and genetic incompatibilities by selfish genetic elements in C. elegans. In chapter 2, I will talk about cichlids bower behaviors where male fishes construct bowers to attract female mates by manipulating sand with their mouths thousands of times over the course of many days. Variations in bower type (‘pits’ and ‘castles’) is one mechanism to create nonrandom mating and maintain a large number of cichlids species in Lake Malawi. To enable quantitative comparisons of these behaviors in different species, an automatic behavior quantification pipeline was built. Specifically, pixel-based Hidden Markov Modeling was combined with density-based spatiotemporal clustering for action detection. Each action video clip was then classified into ten categories using a 3D Residual Network (3D ResNet). These ten categories distinguish spitting, scooping, fin swipes and spawning. I showed that this approach is accurate (> 76% accuracy) in distinguishing fish behaviors and animal intent can be determined from these clips, as spits and scoops performed during bower construction are classified independently from spits and scoops performed during feeding. I applied this approach to >700 hours of video recordings taken from seven independent trials encompassing multiple species and hybrid crosses, collectively containing hundreds of thousands of independent behavioral events. In chapter 3, I use quantitative methods to measure fitness combined with population modeling to study the evolutionary origin of selfish genetic elements and their ability to spread in populations. Previous research found that a toxin-antidote element called peel-zeel is under balancing selection. Here, I explore different models that could cause balancing selection on this locus, which make different predictions on the fitness effect of the peel-zeel locus in hermaphrodites. However, pair-wise competition assays showed the loss of the toxin gene peel-1 decreased fitness of hermaphrodites, contradicting my expectation that peel-1 will decrease animal fitness due to its toxicity. This fitness advantage is independent of the antidote gene zeel-1. This work showed that toxin-antidote systems can spread through populations independent of their selfish effects and suggests linked variants for dauer pheromone response could be responsible for the balancing selection. Finally, in chapter 4, I use simulation methods to study the effect of toxin-antidote elements on linked and unlinked genetic variation in the case of admixture. While both simulation and calculation showed toxin-antidote elements are able to quickly spread in a population without toxin-antidote element, the evolution trajectories of the rest of the genome depends on the initial frequency of the toxin-antidote haplotype in the admixed population. Using calculations and simulations, I showed that unlinked neutral genetic variants will increase their frequency when the initial frequency of peel-zeel is higher than 1/3 and decrease when the initial frequency of peel-zeel is lower than 1/3. My doctoral thesis with many quantitative methods will advance our understanding of the genetic basis of species evolution and evolutional dynamics of selfish genetic elements.Ph.D

Correlations of Genotype with Climate Parameters Suggest Caenorhabditis elegans Niche Adaptations

Species inhabit a variety of environmental niches, and the adaptation to a particular niche is often controlled by genetic factors, including gene-by-environment interactions. The genes that vary in order to regulate the ability to colonize a niche are often difficult to identify, especially in the context of complex ecological systems and in experimentally uncontrolled natural environments. Quantitative genetic approaches provide an opportunity to investigate correlations between genetic factors and environmental parameters that might define a niche. Previously, we have shown how a collection of 208 whole-genome sequenced wild Caenorhabditis elegans can facilitate association mapping approaches. To correlate climate parameters with the variation found in this collection of wild strains, we used geographic data to exhaustively curate daily weather measurements in short-term (3 month), middle-term (one year), and long-term (three year) durations surrounding the date of strain isolation. These climate parameters were used as quantitative traits in association mapping approaches, where we identified 11 quantitative trait loci (QTL) for three climatic variables: elevation, relative humidity, and average temperature. We then narrowed the genomic interval of interest to identify gene candidates with variants potentially underlying phenotypic differences. Additionally, we performed two-strain competition assays at high and low temperatures to validate a QTL that could underlie adaptation to temperature and found suggestive evidence supporting that hypothesis

Selection on a Subunit of the NURF Chromatin Remodeler Modifies Life History Traits in a Domesticated Strain of Caenorhabditis elegans.

Evolutionary life history theory seeks to explain how reproductive and survival traits are shaped by selection through allocations of an individual's resources to competing life functions. Although life-history traits evolve rapidly, little is known about the genetic and cellular mechanisms that control and couple these tradeoffs. Here, we find that two laboratory-adapted strains of C. elegans descended from a single common ancestor that lived in the 1950s have differences in a number of life-history traits, including reproductive timing, lifespan, dauer formation, growth rate, and offspring number. We identified a quantitative trait locus (QTL) of large effect that controls 24%-75% of the total trait variance in reproductive timing at various timepoints. Using CRISPR/Cas9-induced genome editing, we show this QTL is due in part to a 60 bp deletion in the 3' end of the nurf-1 gene, which is orthologous to the human gene encoding the BPTF component of the NURF chromatin remodeling complex. Besides reproduction, nurf-1 also regulates growth rate, lifespan, and dauer formation. The fitness consequences of this deletion are environment specific-it increases fitness in the growth conditions where it was fixed but decreases fitness in alternative laboratory growth conditions. We propose that chromatin remodeling, acting through nurf-1, is a pleiotropic regulator of life history trade-offs underlying the evolution of multiple traits across different species

Preparation and Characterization of Highly Ordered Mercapto-Modified Bridged Silsesquioxane for Removing Ammonia-Nitrogen from Water

In acidic conditions, mesoporous molecular sieves SBA-15 and SBA-15-SH were synthesized. Structural characterization was carried out by powder X-ray diffraction (XRD), Fourier Transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), 13C CP MAS-NMR, 29Si CP MAS-NMR and nitrogen adsorption–desorption (BET). The results showed that in SBA-15-SH, the direct synthesis method made the absorption peak intensity weaker than that of SBA-15, while the post-grafted peak intensity did not change. Their spectra were different due to the C-H stretching bands of Si-O-Si and propyl groups. But their structure was still evenly distributed and was still hexangular mesoporous structure. Their pore size increased, and the H-SBA-15-SH had larger pore size. The adsorption of ammonia-nitrogen by molecular sieve was affected by the relative pressure and the concentration of ammonia-nitrogen, in which the adsorption capacity of G-SBA-15-SH was the largest and the adsorption capacity of SBA-15 was the smallest

A spontaneous complex structural variant in rcan-1 increases exploratory behavior and laboratory fitness of Caenorhabditis elegans.

Over long evolutionary timescales, major changes to the copy number, function, and genomic organization of genes occur, however, our understanding of the individual mutational events responsible for these changes is lacking. In this report, we study the genetic basis of adaptation of two strains of C. elegans to laboratory food sources using competition experiments on a panel of 89 recombinant inbred lines (RIL). Unexpectedly, we identified a single RIL with higher relative fitness than either of the parental strains. This strain also displayed a novel behavioral phenotype, resulting in higher propensity to explore bacterial lawns. Using bulk-segregant analysis and short-read resequencing of this RIL, we mapped the change in exploration behavior to a spontaneous, complex rearrangement of the rcan-1 gene that occurred during construction of the RIL panel. We resolved this rearrangement into five unique tandem inversion/duplications using Oxford Nanopore long-read sequencing. rcan-1 encodes an ortholog to human RCAN1/DSCR1 calcipressin gene, which has been implicated as a causal gene for Down syndrome. The genomic rearrangement in rcan-1 creates two complete and two truncated versions of the rcan-1 coding region, with a variety of modified 5' and 3' non-coding regions. While most copy-number variations (CNVs) are thought to act by increasing expression of duplicated genes, these changes to rcan-1 ultimately result in the reduction of its whole-body expression due to changes in the upstream regions. By backcrossing this rearrangement into a common genetic background to create a near isogenic line (NIL), we demonstrate that both the competitive advantage and exploration behavioral changes are linked to this complex genetic variant. This NIL strain does not phenocopy a strain containing an rcan-1 loss-of-function allele, which suggests that the residual expression of rcan-1 is necessary for its fitness effects. Our results demonstrate how colonization of new environments, such as those encountered in the laboratory, can create evolutionary pressure to modify gene function. This evolutionary mismatch can be resolved by an unexpectedly complex genetic change that simultaneously duplicates and diversifies a gene into two uniquely regulated genes. Our work shows how complex rearrangements can act to modify gene expression in ways besides increased gene dosage

The major-effect QTL on II is partially explained by a 60 bp deletion in <i>nurf-1</i>.

<p><b>a.</b> QTL mapping at four time points plotted on the right arm of chromosome II. Bayesian significance interval is shown as a bar (S.I.). Genetic variants between LSJ2 and N2 plotted along the x-axis (color indicates their location; height of bar has no significance). The boundaries of an introgressed region created surrounding this QTL are shown below the x-axis (<i>kyIR87</i>). <b>b.</b> Egg-laying rate of N2, CX12311, NIL_{<i>nurf-1</i>}, ARL_{<i>nurf-1</i>}, and <i>nurf-1(n4295)</i>. NIL_{<i>nurf-1</i>} is an introgression of LSJ2 DNA near <i>nurf-1</i> (<i>kyIR87</i>, see <b>a</b>) into CX12311. ARL_{<i>nurf-1</i>} is the result of engineering the LSJ2 deletion in <i>nurf-1</i> into the CX12311 background using CRISPR/Cas9. Two independent ARL_{<i>nurf-1</i>} strains were constructed and data show the average of both strains. The <i>nurf-1(n4295)</i> strain was constructed in the N2 background. Black stars (top of triangle) indicate significant differences between N2 and <i>nurf-1(n4295)</i>. Green stars (bottom left) indicate significant differences between CX12311 and NIL_{<i>nurf-1</i>}. Red stars (bottom right) indicate significant differences between CX12311 and ARL_{<i>nurf-1</i>}. Error bars represent S.E.M. <b>c.</b> Genomic region surrounding <i>nurf-1</i> indicating the location of two canonical mutagenesis-derived alleles (<i>n4293</i>, <i>n4295</i>), N2-fixed SNP (WBVar00601361), and LSJ2-fixed 60 bp deletion (WBVar00601565), predicted mRNA and protein product, and predicted effect on protein sequence of the LSJ2 deletion. Isoforms that are predicted to be affected by the WBVar00601565 deletion are colored in light red.</p

Laboratory adaptation of <i>C</i>. <i>elegans</i> strains has resulted in modified reproductive rate and timing.

<p><b>a.</b> History of the <i>C</i>. <i>elegans</i> strains N2 and LSJ2 following isolation from the wild (Bristol, England). LSJ2 was grown in liquid axenic culture whereas N2 was propagated on agar plates. <b>b.</b> Schematic of CX12311 genetic background containing ancestral alleles of <i>npr-1</i> and <i>glb-5</i> backcrossed from CB4856. CB4856 is a wild strain isolated from Hawaii. <b>c.</b> Schematic of egg-laying experiments performed in <b>d</b> and <b>e</b>. <b>d.</b> Averaged egg-laying rate of the CX12311, LSJ2, and <i>daf-22</i> strains starting from the L4 stage. <i>daf-22</i> encodes an enzyme necessary for synthesis of ascaroside pheromones. <b>e.</b> Total number of eggs laid per animal for the three strains from <b>d</b>. Error bars in <b>d</b> and <b>e</b> represent s.e.m.</p