The Genomic Basis of Evolutionary Novelties in a Leafhopper.

Evolutionary innovations generate phenotypic and species diversity. Elucidating the genomic processes underlying such innovations is central to understanding biodiversity. In this study, we addressed the genomic basis of evolutionary novelties in the glassy-winged sharpshooter (Homalodisca vitripennis, GWSS), an agricultural pest. Prominent evolutionary innovations in leafhoppers include brochosomes, proteinaceous structures that are excreted and used to coat the body, and obligate symbiotic associations with two bacterial types that reside within cytoplasm of distinctive cell types. Using PacBio long-read sequencing and Dovetail Omni-C technology, we generated a chromosome-level genome assembly for the GWSS and then validated the assembly using flow cytometry and karyotyping. Additional transcriptomic and proteomic data were used to identify novel genes that underlie brochosome production. We found that brochosome-associated genes include novel gene families that have diversified through tandem duplications. We also identified the locations of genes involved in interactions with bacterial symbionts. Ancestors of the GWSS acquired bacterial genes through horizontal gene transfer (HGT), and these genes appear to contribute to symbiont support. Using a phylogenomics approach, we inferred HGT sources and timing. We found that some HGT events date to the common ancestor of the hemipteran suborder Auchenorrhyncha, representing some of the oldest known examples of HGT in animals. Overall, we show that evolutionary novelties in leafhoppers are generated by the combination of acquiring novel genes, produced both de novo and through tandem duplication, acquiring new symbiotic associations that enable use of novel diets and niches, and recruiting foreign genes to support symbionts and enhance herbivory.

Comparison of cytotoxic potency between freshly cultured and freshly thawed cytokine-induced killer cells from human umbilical cord blood.

Immune cell therapy has been incorporated into cancer therapy over the past few years. Chimeric antigen receptor T cells (Car-T cells) transplantation is a novel and promising therapy for cancer treatment and introduces a new age of immune cell therapy. However, the expensive nature of genetic modification procedures limits the accessibility of Car-T cells for cancer treatment. Cytokine-induced killer cells (CIKs) can kill the target cells in an MHC-non-restricted manner; these cells can be developed to "off-the-shelf" immune cell products for cancer treatment. However, the anti-tumor potency of freshly thawed CIKs is not well documented. This study aimed to fill this gap, evaluating the anti-tumor potency of freshly thawed CIKs compared to that of freshly cultured CIKs. CIKs were produced from the human umbilical cord blood in accordance with published protocols. CIKs were cryopreserved in xeno-free cryomedium that contains 5% DMSO, 10% human serum in phosphate buffer saline at - 86 °C. These cells were thawed and immediately utilized in assays (called freshly thawed CIKs) with freshly cultured cells are control. The expression of the surface markers of CIKs, cytokine production, and in vitro anti-tumor cytotoxic cells of freshly thawed CIKs were evaluated and compared to freshly cultured CIKs. Additionally, the freshly thawed CIKs were injected into the breast of tumor-bearing mice to assess the anti-tumor potency in vivo. The results obtained in freshly thawed CIKs and freshly cultured CIKs demonstrated that the expression of CD3, and CD56 were comparable in both cases. The production of TNF-α, IFN-γ, and IL-10 was slightly reduced in freshly thawed cells compared to the freshly cultured cells. The in vitro lysis toward MCF-7 cancer cells was similar between freshly thawed and freshly cultured CIKs. Moreover, the freshly thawed CIKs displayed anti-breast tumor activity in the breast tumor-bearing mice. The volume of tumors significantly reduced in the mice grafted with freshly thawed CIKs while, conversely, the tumor volume in mice of the placebo group gradually increased. This study substantiated that freshly thawed CIKs preserved their anti-tumor potency in both in vitro and in vivo conditions. The results initially revealed the great potential of UCB-CIKs for "off-the-shelf" CIK product manufacturing. However, further studies on the effects of cryomedia, freezing rate, and thawing procedure should be undertaken before freshly thawed off-the-shelf UCB-CIKs are utilized in clinical trials.

Long-read sequence and assembly of segmental duplications.

We have developed a computational method based on polyploid phasing of long sequence reads to resolve collapsed regions of segmental duplications within genome assemblies. Segmental Duplication Assembler (SDA; https://github.com/mvollger/SDA ) constructs graphs in which paralogous sequence variants define the nodes and long-read sequences provide attraction and repulsion edges, enabling the partition and assembly of long reads corresponding to distinct paralogs. We apply it to single-molecule, real-time sequence data from three human genomes and recover 33-79 megabase pairs (Mb) of duplications in which approximately half of the loci are diverged (<99.8%) compared to the reference genome. We show that the corresponding sequence is highly accurate (>99.9%) and that the diverged sequence corresponds to copy-number-variable paralogs that are absent from the human reference genome. Our method can be applied to other complex genomes to resolve the last gene-rich gaps, improve duplicate gene annotation, and better understand copy-number-variant genetic diversity at the base-pair level.

A systematic, label-free method for identifying RNA-associated proteins in vivo provides insights into vertebrate ciliary beating machinery.

Cell-type specific RNA-associated proteins are essential for development and homeostasis in animals. Despite a massive recent effort to systematically identify RNA-associated proteins, we currently have few comprehensive rosters of cell-type specific RNA-associated proteins in vertebrate tissues. Here, we demonstrate the feasibility of determining the RNA-associated proteome of a defined vertebrate embryonic tissue using DIF-FRAC, a systematic and universal (i.e., label-free) method. Application of DIF-FRAC to cultured tissue explants of Xenopus mucociliary epithelium identified dozens of known RNA-associated proteins as expected, but also several novel RNA-associated proteins, including proteins related to assembly of the mitotic spindle and regulation of ciliary beating. In particular, we show that the inner dynein arm tether Cfap44 is an RNA-associated protein that localizes not only to axonemes, but also to liquid-like organelles in the cytoplasm called DynAPs. This result led us to discover that DynAPs are generally enriched for RNA. Together, these data provide a useful resource for a deeper understanding of mucociliary epithelia and demonstrate that DIF-FRAC will be broadly applicable for systematic identification of RNA-associated proteins from embryonic tissues.

Recurrent structural variation, clustered sites of selection, and disease risk for the complement factor H (CFH) gene family.

Structural variation and single-nucleotide variation of the complement factor H (CFH) gene family underlie several complex genetic diseases, including age-related macular degeneration (AMD) and atypical hemolytic uremic syndrome (AHUS). To understand its diversity and evolution, we performed high-quality sequencing of this ∼360-kbp locus in six primate lineages, including multiple human haplotypes. Comparative sequence analyses reveal two distinct periods of gene duplication leading to the emergence of four CFH-related (CFHR) gene paralogs (CFHR2 and CFHR4 ∼25-35 Mya and CFHR1 and CFHR3 ∼7-13 Mya). Remarkably, all evolutionary breakpoints share a common ∼4.8-kbp segment corresponding to an ancestral CFHR gene promoter that has expanded independently throughout primate evolution. This segment is recurrently reused and juxtaposed with a donor duplication containing exons 8 and 9 from ancestral CFH, creating four CFHR fusion genes that include lineage-specific members of the gene family. Combined analysis of >5,000 AMD cases and controls identifies a significant burden of a rare missense mutation that clusters at the N terminus of CFH [P = 5.81 × 10-8, odds ratio (OR) = 9.8 (3.67-Infinity)]. A bipolar clustering pattern of rare nonsynonymous mutations in patients with AMD (P < 10-3) and AHUS (P = 0.0079) maps to functional domains that show evidence of positive selection during primate evolution. Our structural variation analysis in >2,400 individuals reveals five recurrent rearrangement breakpoints that show variable frequency among AMD cases and controls. These data suggest a dynamic and recurrent pattern of mutation critical to the emergence of new CFHR genes but also in the predisposition to complex human genetic disease phenotypes.

Prader-Willi Syndrome due to an Unbalanced de novo Translocation t(15;19)(q12;p13.3).

Prader-Willi syndrome (PWS) is a complex, multisystem genetic disorder characterized by endocrine, neurologic, and behavioral abnormalities. We report the first case of an unbalanced de novo reciprocal translocation of chromosomes 15 and 19, 45,XY,-15,der(19)t(15;19)(q12;p13.3), resulting in monosomy for the PWS critical chromosome region. Our patient had several typical features of PWS including infantile hypotonia, a poor suck and feeding difficulties, tantrums, skin picking, compulsions, small hands and feet, and food seeking, but not hypopigmentation, a micropenis, cryptorchidism or obesity as common findings seen in PWS at the time of examination at 6 years of age. He had seizures noted from 1 to 3 years of age and marked cognitive delay. High-resolution SNP microarray analysis identified an atypical PWS type I deletion in chromosome 15 involving the proximal breakpoint BP1. The deletion extended beyond the GABRB3 gene but was proximal to the usual distal breakpoint (BP3) within the 15q11q13 region, and GABRA5, GABRG3, and OCA2 genes were intact. No deletion of band 19p13.3 was detected; therefore, the patient was not at an increased risk of tumors from the Peutz-Jeghers syndrome associated with a deletion of the STK11 gene.

Systematic profiling of ale yeast protein dynamics across fermentation and repitching.

Studying the genetic and molecular characteristics of brewing yeast strains is crucial for understanding their domestication history and adaptations accumulated over time in fermentation environments, and for guiding optimizations to the brewing process itself. Saccharomyces cerevisiae (brewing yeast) is among the most profiled organisms on the planet, yet the temporal molecular changes that underlie industrial fermentation and beer brewing remain understudied. Here, we characterized the genomic makeup of a Saccharomyces cerevisiae ale yeast widely used in the production of Hefeweizen beers, and applied shotgun mass spectrometry to systematically measure the proteomic changes throughout 2 fermentation cycles which were separated by 14 rounds of serial repitching. The resulting brewing yeast proteomics resource includes 64,740 protein abundance measurements. We found that this strain possesses typical genetic characteristics of Saccharomyces cerevisiae ale strains and displayed progressive shifts in molecular processes during fermentation based on protein abundance changes. We observed protein abundance differences between early fermentation batches compared to those separated by 14 rounds of serial repitching. The observed abundance differences occurred mainly in proteins involved in the metabolism of ergosterol and isobutyraldehyde. Our systematic profiling serves as a starting point for deeper characterization of how the yeast proteome changes during commercial fermentations and additionally serves as a resource to guide fermentation protocols, strain handling, and engineering practices in commercial brewing and fermentation environments. Finally, we created a web interface (https://brewing-yeast-proteomics.ccbb.utexas.edu/) to serve as a valuable resource for yeast geneticists, brewers, and biochemists to provide insights into the global trends underlying commercial beer production.

Houston Methodist Ruptured Abdominal Aortic Aneurysm Guidelines.

Ruptured abdominal aortic aneurysm (RAAA) is an acute aortic condition that requires emergent intervention and appropriate continuity of care to optimize patient outcomes. We describe the standardized RAAA protocol at the Houston Methodist Hospital Acute Aortic Treatment Center, developed to navigate critical patient transfer periods safely and efficiently, make crucial decisions about surgical intervention, and clearly communicate these plans with other care team providers. Our workflow is organized into five phases: prehospital, preoperative, intraoperative, postoperative, and post-discharge. We identify the transfer center, anesthesia, operating room nursing staff, surgeons, and intensive care unit as key entities of our acute aortic pathology care team. This systematic protocol for the management of acute aortic emergencies such as RAAA identifies critical decision points, potential complications at each stage, and recommendations for best practice.

Cadaveric aortic aneurysm creation: A life-like model for training endovascular repair.

The recent decline in RAAA incidence and the fast paced scenario with associated challenges regarding training calls for initiative for a better training environment to maximize learning. This led us to the creation of a pulsatile human cadaveric RAAA model. Fresh frozen cadaver was used to create RAAA with BioTissue in hybrid suite with ability to perform CBCTA for sizing. As a proof of concept, the model was used to perform REVAR with proximal CODA balloon control. The model proved to be feasible and we believe it is a better environment to train and gain adequate proficiency in RAAA management.

Systematic Profiling of Ale Yeast Protein Dynamics across Fermentation and Repitching.

Studying the genetic and molecular characteristics of brewing yeast strains is crucial for understanding their domestication history and adaptations accumulated over time in fermentation environments, and for guiding optimizations to the brewing process itself. Saccharomyces cerevisiae (brewing yeast) is amongst the most profiled organisms on the planet, yet the temporal molecular changes that underlie industrial fermentation and beer brewing remain understudied. Here, we characterized the genomic makeup of a Saccharomyces cerevisiae ale yeast widely used in the production of Hefeweizen beers, and applied shotgun mass spectrometry to systematically measure the proteomic changes throughout two fermentation cycles which were separated by 14 rounds of serial repitching. The resulting brewing yeast proteomics resource includes 64,740 protein abundance measurements. We found that this strain possesses typical genetic characteristics of Saccharomyces cerevisiae ale strains and displayed progressive shifts in molecular processes during fermentation based on protein abundance changes. We observed protein abundance differences between early fermentation batches compared to those separated by 14 rounds of serial repitching. The observed abundance differences occurred mainly in proteins involved in the metabolism of ergosterol and isobutyraldehyde. Our systematic profiling serves as a starting point for deeper characterization of how the yeast proteome changes during commercial fermentations and additionally serves as a resource to guide fermentation protocols, strain handling, and engineering practices in commercial brewing and fermentation environments. Finally, we created a web interface (https://brewing-yeast-proteomics.ccbb.utexas.edu/) to serve as a valuable resource for yeast geneticists, brewers, and biochemists to provide insights into the global trends underlying commercial beer production.

Co-fractionation/mass spectrometry to identify protein complexes.

Co-fractionation/mass spectrometry (CF/MS) is a flexible and powerful method to detect physical associations of proteins. CF/MS can be applied to any tissue or organism without the need for protein-specific antibodies or epitope tags. Here, we outline two alternate protocols for MS preparation of samples (containing low or high salt) and a computational pipeline (cfmsflow) that together allow the successful application of this approach. These protocols are based on CF/MS of over 16 diverse organisms including plants and animals. For complete details on the use and execution of this protocol, please refer to McWhite et al. (2020).

Evidence for opposing selective forces operating on human-specific duplicated TCAF genes in Neanderthals and humans.

TRP channel-associated factor 1/2 (TCAF1/TCAF2) proteins antagonistically regulate the cold-sensor protein TRPM8 in multiple human tissues. Understanding their significance has been complicated given the locus spans a gap-ridden region with complex segmental duplications in GRCh38. Using long-read sequencing, we sequence-resolve the locus, annotate full-length TCAF models in primate genomes, and show substantial human-specific TCAF copy number variation. We identify two human super haplogroups, H4 and H5, and establish that TCAF duplications originated ~1.7 million years ago but diversified only in Homo sapiens by recurrent structural mutations. Conversely, in all archaic-hominin samples the fixation for a specific H4 haplotype without duplication is likely due to positive selection. Here, our results of TCAF copy number expansion, selection signals in hominins, and differential TCAF2 expression between haplogroups and high TCAF2 and TRPM8 expression in liver and prostate in modern-day humans imply TCAF diversification among hominins potentially in response to cold or dietary adaptations.

Tobacco mosaic virus movement protein enhances the spread of RNA silencing.

Eukaryotic cells restrain the activity of foreign genetic elements, including viruses, through RNA silencing. Although viruses encode suppressors of silencing to support their propagation, viruses may also exploit silencing to regulate host gene expression or to control the level of their accumulation and thus to reduce damage to the host. RNA silencing in plants propagates from cell to cell and systemically via a sequence-specific signal. Since the signal spreads between cells through plasmodesmata like the viruses themselves, virus-encoded plasmodesmata-manipulating movement proteins (MP) may have a central role in compatible virus:host interactions by suppressing or enhancing the spread of the signal. Here, we have addressed the propagation of GFP silencing in the presence and absence of MP and MP mutants. We show that the protein enhances the spread of silencing. Small RNA analysis indicates that MP does not enhance the silencing pathway but rather enhances the transport of the signal through plasmodesmata. The ability to enhance the spread of silencing is maintained by certain MP mutants that can move between cells but which have defects in subcellular localization and do not support the spread of viral RNA. Using MP expressing and non-expressing virus mutants with a disabled silencing suppressing function, we provide evidence indicating that viral MP contributes to anti-viral silencing during infection. Our results suggest a role of MP in controlling virus propagation in the infected host by supporting the spread of silencing signal. This activity of MP involves only a subset of its properties implicated in the spread of viral RNA.

Adaptive archaic introgression of copy number variants and the discovery of previously unknown human genes.

Copy number variants (CNVs) are subject to stronger selective pressure than single-nucleotide variants, but their roles in archaic introgression and adaptation have not been systematically investigated. We show that stratified CNVs are significantly associated with signatures of positive selection in Melanesians and provide evidence for adaptive introgression of large CNVs at chromosomes 16p11.2 and 8p21.3 from Denisovans and Neanderthals, respectively. Using long-read sequence data, we reconstruct the structure and complex evolutionary history of these polymorphisms and show that both encode positively selected genes absent from most human populations. Our results collectively suggest that large CNVs originating in archaic hominins and introgressed into modern humans have played an important role in local population adaptation and represent an insufficiently studied source of large-scale genetic variation.

Modification of small RNAs associated with suppression of RNA silencing by tobamovirus replicase protein.

Plant viruses act as triggers and targets of RNA silencing and have evolved proteins to suppress this plant defense response during infection. Although Tobacco mosaic tobamovirus (TMV) triggers the production of virus-specific small interfering RNAs (siRNAs), this does not lead to efficient silencing of TMV nor is a TMV-green fluorescent protein (GFP) hybrid able to induce silencing of a GFP-transgene in Nicotiana benthamiana, indicating that a TMV silencing suppressor is active and acts downstream of siRNA production. On the other hand, TMV-GFP is unable to spread into cells in which GFP silencing is established, suggesting that the viral silencing suppressor cannot revert silencing that is already established. Although previous evidence indicates that the tobamovirus silencing suppressing activity resides in the viral 126-kDa small replicase subunit, the mechanism of silencing suppression by this virus family is not known. Here, we connect the silencing suppressing activity of this protein with our previous finding that Oilseed rape mosaic tobamovirus infection leads to interference with HEN1-mediated methylation of siRNA and micro-RNA (miRNA). We demonstrate that TMV infection similarly leads to interference with HEN1-mediated methylation of small RNAs and that this interference and the formation of virus-induced disease symptoms are linked to the silencing suppressor activity of the 126-kDa protein. Moreover, we show that also Turnip crinkle virus interferes with the methylation of siRNA but, in contrast to tobamoviruses, not with the methylation of miRNA.