Integrative Analysis of Germline Rare Variants in Clear and Non-clear Cell Renal Cell Carcinoma.

Background and objective: Previous germline studies on renal cell carcinoma (RCC) have usually pooled clear and non-clear cell RCCs and have not adequately accounted for population stratification, which might have led to an inaccurate estimation of genetic risk. Here, we aim to analyze the major germline drivers of RCC risk and clinically relevant but underexplored germline variant types. Methods: We first characterized germline pathogenic variants (PVs), cryptic splice variants, and copy number variants (CNVs) in 1436 unselected RCC patients. To evaluate the enrichment of PVs in RCC, we conducted a case-control study of 1356 RCC patients ancestry matched with 16 512 cancer-free controls using approaches accounting for population stratification and histological subtypes, followed by characterization of secondary somatic events. Key findings and limitations: Clear cell RCC patients (n = 976) exhibited a significant burden of PVs in VHL compared with controls (odds ratio [OR]: 39.1, p = 4.95e-05). Non-clear cell RCC patients (n = 380) carried enrichment of PVs in FH (OR: 77.9, p = 1.55e-08) and MET (OR: 1.98e11, p = 2.07e-05). In a CHEK2-focused analysis with European participants, clear cell RCC (n = 906) harbored nominal enrichment of low-penetrance CHEK2 variants-p.Ile157Thr (OR: 1.84, p = 0.049) and p.Ser428Phe (OR: 5.20, p = 0.045), while non-clear cell RCC (n = 295) exhibited nominal enrichment of CHEK2 loss of function PVs (OR: 3.51, p = 0.033). Patients with germline PVs in FH, MET, and VHL exhibited significantly earlier age of cancer onset than patients without germline PVs (mean: 46.0 vs 60.2 yr, p < 0.0001), and more than half had secondary somatic events affecting the same gene (n = 10/15, 66.7%). Conversely, CHEK2 PV carriers exhibited a similar age of onset to patients without germline PVs (mean: 60.1 vs 60.2 yr, p = 0.99), and only 30.4% carried somatic events in CHEK2 (n = 7/23). Finally, pathogenic germline cryptic splice variants were identified in SDHA and TSC1, and pathogenic germline CNVs were found in 18 patients, including CNVs in FH, SDHA, and VHL. Conclusions and clinical implications: This analysis supports the existing link between several RCC risk genes and RCC risk manifesting in earlier age of onset. It calls for caution when assessing the role of CHEK2 due to the burden of founder variants with varying population frequency. It also broadens the definition of the RCC germline landscape of pathogenicity to incorporate previously understudied types of germline variants. Patient summary: In this study, we carefully compared the frequency of rare inherited mutations with a focus on patients' genetic ancestry. We discovered that subtle variations in genetic background may confound a case-control analysis, especially in evaluating the cancer risk associated with specific genes, such as CHEK2. We also identified previously less explored forms of rare inherited mutations, which could potentially increase the risk of kidney cancer.

Historical perspective and future directions: computational science in immuno-oncology.

Immuno-oncology holds promise for transforming patient care having achieved durable clinical response rates across a variety of advanced and metastatic cancers. Despite these achievements, only a minority of patients respond to immunotherapy, underscoring the importance of elucidating molecular mechanisms responsible for response and resistance to inform the development and selection of treatments. Breakthroughs in molecular sequencing technologies have led to the generation of an immense amount of genomic and transcriptomic sequencing data that can be mined to uncover complex tumor-immune interactions using computational tools. In this review, we discuss existing and emerging computational methods that contextualize the composition and functional state of the tumor microenvironment, infer the reactivity and clonal dynamics from reconstructed immune cell receptor repertoires, and predict the antigenic landscape for immune cell recognition. We further describe the advantage of multi-omics analyses for capturing multidimensional relationships and artificial intelligence techniques for integrating omics data with histopathological and radiological images to encapsulate patterns of treatment response and tumor-immune biology. Finally, we discuss key challenges impeding their widespread use and clinical application and conclude with future perspectives. We are hopeful that this review will both serve as a guide for prospective researchers seeking to use existing tools for scientific discoveries and inspire the optimization or development of novel tools to enhance precision, ultimately expediting advancements in immunotherapy that improve patient survival and quality of life.

Sin Nombre Virus and the Emergence of Other Hantaviruses: A Review of the Biology, Ecology, and Disease of a Zoonotic Pathogen.

Sin Nombre virus (SNV) is an emerging virus that was first discovered in the Four Corners region of the United States in 1993. The virus causes a disease known as Hantavirus Pulmonary Syndrome (HPS), sometimes called Hantavirus Cardiopulmonary Syndrome (HCPS), a life-threatening illness named for the predominance of infection of pulmonary endothelial cells. SNV is one of several rodent-borne hantaviruses found in the western hemisphere with the capability of causing this disease. The primary reservoir of SNV is the deer mouse (Peromyscus maniculatus), and the virus is transmitted primarily through aerosolized rodent excreta and secreta. Here, we review the history of SNV emergence and its virus biology and relationship to other New World hantaviruses, disease, treatment, and prevention options.

Colossal C130 Fullertubes: Soluble [5,5] C130-D5h(1) Pristine Molecules with 70 Nanotube Carbons and Two 30-Atom Hemifullerene End-caps.

We report the seminal experimental isolation and DFT characterization of pristine [5,5] C130-D5h(1) fullertubes. This achievement represents the largest soluble carbon molecule obtained in its pristine form. The [5,5] C130 species is the highest aspect ratio fullertube purified to date and now surpasses the recent gigantic [5,5] C120-D5d(1). In contrast to C90, C100, and C120 fullertubes, the longer C130-D5h has more nanotubular carbons (70) than end-cap fullerenyl atoms (60). Starting from 39,393 possible C130 isolated pentagon rule (IPR) structures and after analyzing polarizability, retention time, and UV-vis spectra, these three layers of data remarkably predict a single candidate isomer and fullertube, [5,5] C130-D5h(1). This structural assignment is augmented by atomic resolution STEM data showing distinctive and tubular "pill-like" structures with diameters and aspect ratios consistent with [5,5] C130-D5h(1) fullertubes. The high selectivity of the aminopropanol reaction with spheroidal fullerenes permits facile separation and removal of fullertubes from soot extracts. Experimental analyses (HPLC retention time, UV-vis, and STEM) were synergistically used (with polarizability and DFT property calculations) to down select and confirm the C130 fullertube structure. Achieving the isolation of a new [5,5] C130-D5h fullertube opens the door to application development and fundamental studies of electron confinement, fluorescence, and metallic character for a fullertube series of molecules with systematic tubular elongation. This [5,5] fullertube family also invites comparative studies with single-walled carbon nanotubes (SWCNTs), nanohorns (SWCNHs), and fullerenes.

Structure-activity relationship of dihydropyridines for rhabdomyosarcoma.

Childhood muscle-related cancer rhabdomyosarcoma is a rare disease with a 50-year unmet clinical need for the patients presented with advanced disease. The rarity of ∼350 cases per year in North America generally diminishes the viability of large-scale, pharmaceutical industry driven drug development efforts for rhabdomyosarcoma. In this study, we performed a large-scale screen of 640,000 compounds to identify the dihydropyridine (DHP) class of anti-hypertensives as a priority compound hit. A structure-activity relationship was uncovered with increasing cell growth inhibition as side chain length increases at the ortho and para positions of the parent DHP molecule. Growth inhibition was consistent across n = 21 rhabdomyosarcoma cell line models. Anti-tumor activity in vitro was paralleled by studies in vivo. The unexpected finding was that the action of DHPs appears to be other than on the DHP receptor (i.e., L-type voltage-gated calcium channel). These findings provide the basis of a medicinal chemistry program to develop dihydropyridine derivatives that retain anti-rhabdomyosarcoma activity without anti-hypertensive effects.

The hydroxyproline O-arabinosyltransferase FIN4 is required for tomato pollen intine development.

The pollen grain cell wall is a highly specialized structure composed of distinct layers formed through complex developmental pathways. The production of the innermost intine layer, composed of cellulose, pectin and other polymers, is particularly poorly understood. Here we demonstrate an important and specific role for the hydroxyproline O-arabinosyltransferase (HPAT) FIN4 in tomato intine development. HPATs are plant-specific enzymes which initiate glycosylation of certain cell wall structural proteins and signaling peptides. FIN4 was expressed throughout pollen development in both the developing pollen and surrounding tapetal cells. A fin4 mutant with a partial deletion of the catalytic domain displayed significantly reduced male fertility in vivo and compromised pollen hydration and germination in vitro. However, fin4 pollen that successfully germinated formed morphologically normal pollen tubes with the same growth rate as the wild-type pollen. When we examined mature fin4 pollen, we found they were cytologically normal, and formed morphologically normal exine, but produced significantly thinner intine. During intine deposition at the late stages of pollen development we found fin4 pollen had altered polymer deposition, including reduced cellulose and increased detection of pectin, specifically homogalacturonan with both low and high degrees of methylesterification. Therefore, FIN4 plays an important role in intine formation and, in turn pollen hydration and germination and the process of intine formation involves dynamic changes in the developing pollen cell wall.

Integrative Analysis of Germline Rare Variants in Clear and Non-Clear Cell Renal Cell Carcinoma.

IMPORTANCE: RCC encompasses a set of histologically distinct cancers with a high estimated genetic heritability, of which only a portion is currently explained. Previous rare germline variant studies in RCC have usually pooled clear and non-clear cell RCCs and have not adequately accounted for population stratification that may significantly impact the interpretation and discovery of certain candidate risk genes. OBJECTIVE: To evaluate the enrichment of germline PVs in established cancer-predisposing genes (CPGs) in clear cell and non-clear cell RCC patients compared to cancer-free controls using approaches that account for population stratification and to identify unconventional types of germline RCC risk variants that confer an increased risk of developing RCC. DESIGN SETTING AND PARTICIPANTS: In 1,436 unselected RCC patients with sufficient data quality, we systematically identified rare germline PVs, cryptic splice variants, and copy number variants (CNVs). From this unselected cohort, 1,356 patients were ancestry-matched with 16,512 cancer-free controls, and gene-level enrichment of rare germline PVs were assessed in 143 CPGs, followed by an investigation of somatic events in matching tumor samples. MAIN OUTCOMES AND MEASURES: Gene-level burden of rare germline PVs, identification of secondary somatic events accompanying the germline PVs, and characterization of less-explored types of rare germline PVs in RCC patients. RESULTS: In clear cell RCC (n = 976 patients), patients exhibited significantly higher prevalence of PVs in VHL compared to controls (OR: 39.1, 95% CI: 7.01-218.07, p-value:4.95e-05, q-value:0.00584). In non-clear cell RCC (n = 380 patients), patients carried enriched burden of PVs in FH (OR: 77.9, 95% CI: 18.68-324.97, p-value:1.55e-08, q-value: 1.83e-06) and MET (OR: 1.98e11, 95% CI: 0-inf, p-value: 2.07e-05, q-value: 3.50e-07). In a CHEK2-focused analysis with European cases and controls, clear cell RCC patients (n=906 European patients) harbored nominal enrichment of the previously reported low-penetrance CHEK2 variants, p.Ile157Thr (OR:1.84, 95% CI: 1.00-3.36, p-value:0.049) and p.Ser428Phe (OR:5.20, 95% CI: 1.00-26.40, p-value:0.045) while non-clear cell RCC patients (n=295 European patients) exhibited nominal enrichment of CHEK2 LOF germline PVs (OR: 3.51, 95% CI: 1.10-11.10, p-value: 0.033). RCC patients with germline PVs in FH, MET, and VHL exhibited significantly earlier age of cancer onset compared to patients without any germline PVs in CPGs (Mean: 46.0 vs 60.2 years old, Tukey adjusted p-value < 0.0001), and more than half had secondary somatic events affecting the same gene (n=10/15, 66.7%, 95% CI: 38.7-87.0%). Conversely, patients with rare germline PVs in CHEK2 exhibited a similar age of disease onset to patients without any identified germline PVs in CPGs (Mean: 60.1 vs 60.2 years old, Tukey adjusted p-value: 0.99), and only 30.4% of the patients carried secondary somatic events in CHEK2 (n=7/23, 95% CI: 14.1-53.0%). Finally, rare pathogenic germline cryptic splice variants underexplored in RCC were identified in SDHA and TSC1, and rare pathogenic germline CNVs were found in 18 patients, including CNVs in FH, SDHA, and VHL. CONCLUSIONS AND RELEVANCE: This systematic analysis supports the existing link between several RCC risk genes and elevated RCC risk manifesting in earlier age of RCC onset. Our analysis calls for caution when assessing the role of germline PVs in CHEK2 due to the burden of founder variants with varying population frequency in different ancestry groups. It also broadens the definition of the RCC germline landscape of pathogenicity to incorporate previously understudied types of germline variants, such as cryptic splice variants and CNVs.

Emergency embolization of a massive life-threatening placenta percreta bleeding following delivery by Cesarean section.

The rise in the number of Cesarean sections (CS) worldwide has increased the incidence of the placenta accreta spectrum disorders in the past years. About 5% of patients undergoing a CS develop placenta percreta. A 30-year-old woman, G2P1 with previous uncomplicated CS delivery had an elective CS delivery at 37w6d. The delivery was complicated by a substantial hemorrhage. On emergency laparotomy a placenta percreta was seen in the broad ligament, which could not be removed surgically. Embolization was performed with Gelfoam particles until stasis in the right uterine artery with placement of a coil. Patient discharge was 12 days after intervention. Emergency embolization is an effective treatment in bleeding complications due to placenta percreta at partus.

Gigantic C120 Fullertubes: Prediction and Experimental Evidence for Isomerically Purified Metallic [5,5] C120-D5d(1) and Nonmetallic [10,0] C120-D5h(10766).

We report the first experimental characterization of isomerically pure and pristine C120 fullertubes, [5,5] C120-D5d(1) and [10,0] C120-D5h(10766). These new molecules represent the highest aspect ratio fullertubes isolated to date; for example, the prior largest empty cage fullertube was [5,5] C100-D5d(1). This increase of 20 carbon atoms represents a gigantic leap in comparison to three decades of C60-C90 fullerene research. Moreover, the [10,0] C120-D5d(10766) fullertube has an end-cap derived from C80-Ih and is a new fullertube whose C40 end-cap has not yet been isolated experimentally. Theoretical and experimental analyses of anisotropic polarizability and UV-vis assign C120 isomer I as a [5,5] C120-D5d(1) fullertube. C120 isomer II matches a [10,0] C120-D5h(10766) fullertube. These structural assignments are further supported by Raman data showing metallic character for [5,5] C120-D5d(1) and nonmetallic character for C120-D5h(10766). STM imaging reveals a tubular structure with an aspect ratio consistent with a [5,5] C120-D5d(1) fullertube. With microgram quantities not amenable to crystallography, we demonstrate that DFT anisotropic polarizability, augmented by long-accepted experimental analyses (HPLC retention time, UV-vis, Raman, and STM) can be synergistically used (with DFT) to down select, predict, and assign C120 fullertube candidate structures. From 10 774 mathematically possible IPR C120 structures, this anisotropic polarizability paradigm is quite favorable to distinguish tubular structures from carbon soot. Identification of isomers I and II was surprisingly facile, i.e., two purified isomers for two possible structures of widely distinguishing features. These metallic and nonmetallic C120 fullertube isomers open the door to both fundamental research and application development.

Tissue-resident memory and circulating T cells are early responders to pre-surgical cancer immunotherapy.

Neoadjuvant immune checkpoint blockade has shown promising clinical activity. Here, we characterized early kinetics in tumor-infiltrating and circulating immune cells in oral cancer patients treated with neoadjuvant anti-PD-1 or anti-PD-1/CTLA-4 in a clinical trial (NCT02919683). Tumor-infiltrating CD8 T cells that clonally expanded during immunotherapy expressed elevated tissue-resident memory and cytotoxicity programs, which were already active prior to therapy, supporting the capacity for rapid response. Systematic target discovery revealed that treatment-expanded tumor T cell clones in responding patients recognized several self-antigens, including the cancer-specific antigen MAGEA1. Treatment also induced a systemic immune response characterized by expansion of activated T cells enriched for tumor-infiltrating T cell clonotypes, including both pre-existing and emergent clonotypes undetectable prior to therapy. The frequency of activated blood CD8 T cells, notably pre-treatment PD-1-positive KLRG1-negative T cells, was strongly associated with intra-tumoral pathological response. These results demonstrate how neoadjuvant checkpoint blockade induces local and systemic tumor immunity.

O-glycosylation of the extracellular domain of pollen class I formins modulates their plasma membrane mobility.

In plant cells, linkage between the cytoskeleton, plasma membrane, and cell wall is crucial for maintaining cell shape. In highly polarized pollen tubes, this coordination is especially important to allow rapid tip growth and successful fertilization. Class I formins contain cytoplasmic actin-nucleating formin homology domains as well as a proline-rich extracellular domain and are candidate coordination factors. Here, using Arabidopsis, we investigated the functional significance of the extracellular domain of two pollen-expressed class I formins: AtFH3, which does not have a polar localization, and AtFH5, which is limited to the growing tip region. We show that the extracellular domain of both is necessary for their function, and identify distinct O-glycans attached to these sequences, AtFH5 being hydroxyproline-arabinosylated and AtFH3 carrying arabinogalactan chains. Loss of hydroxyproline arabinosylation altered the plasma membrane localization of AtFH5 and disrupted actin cytoskeleton organization. Moreover, we show that O-glycans differentially affect lateral mobility in the plasma membrane. Together, our results support a model of protein sub-functionalization in which AtFH5 and AtFH3, restricted to specific plasma membrane domains by their extracellular domains and the glycans attached to them, organize distinct subarrays of actin during pollen tube elongation.

SEC1A is a major Arabidopsis Sec1/Munc18 gene in vesicle trafficking during pollen tube tip growth.

Pollen tubes (PTs) grow by the targeted secretion of new cell wall material to their expanding tip region. Sec1/Munc18 (SM) proteins promote membrane fusion through regulation of the SNARE complex. We have previously shown that disruption of protein glycosylation in the Arabidopsis thaliana hpat1 hpat3 double mutant leads to PT growth defects that can be suppressed by reducing secretion. Here, we identified five point mutant alleles of the SM protein SEC1A as hpat1/3 suppressors. The suppressors increased seed set, reduced PT growth defects and reduced the rate of glycoprotein secretion. In the absence of the hpat mutations, sec1a reduced pollen germination and PT elongation producing shorter and wider PTs. Consistent with a defect in membrane fusion, sec1a PTs accumulated secretory vesicles. Though sec1a had significantly reduced male transmission, homozygous sec1a plants maintained full seed set, demonstrating that SEC1A was ultimately dispensable for pollen fertility. However, when combined with a mutation in another SEC1-like SM gene, keule, pollen fertility was totally abolished. Mutation in sec1b, the final member of the Arabidopsis SEC1 clade, did not enhance the sec1a phenotype. Thus, SEC1A is the major SM protein promoting pollen germination and tube elongation, but in its absence KEULE can partially supply this activity. When we examined the expression of the SM protein family in other species for which pollen expression data were available, we found that at least one Sec1-like protein was highly expressed in pollen samples, suggesting a conserved role in pollen fertility in other species.

Sequential Deposition and Remodeling of Cell Wall Polymers During Tomato Pollen Development.

The cell wall of a mature pollen grain is a highly specialized, multilayered structure. The outer, sporopollenin-based exine provides protection and support to the pollen grain, while the inner intine, composed primarily of cellulose, is important for pollen germination. The formation of the mature pollen grain wall takes place within the anther with contributions of cell wall material from both the developing pollen grain as well as the surrounding cells of the tapetum. The process of wall development is complex; multiple cell wall polymers are deposited, some transiently, in a controlled sequence of events. Tomato (Solanum lycopersicum) is an important agricultural crop, which requires successful fertilization for fruit production as do many other members of the Solanaceae family. Despite the importance of pollen development for tomato, little is known about the detailed pollen gain wall developmental process. Here, we describe the structure of the tomato pollen wall and establish a developmental timeline of its formation. Mature tomato pollen is released from the anther in a dehydrated state and is tricolpate, with three long apertures without overlaying exine from which the pollen tube may emerge. Using histology and immunostaining, we determined the order in which key cell wall polymers were deposited with respect to overall pollen and anther development. Pollen development began in young flower buds when the premeiotic microspore mother cells (MMCs) began losing their cellulose primary cell wall. Following meiosis, the still conjoined microspores progressed to the tetrad stage characterized by a temporary, thick callose wall. Breakdown of the callose wall released the individual early microspores. Exine deposition began with the secretion of the sporopollenin foot layer. At the late microspore stage, exine deposition was completed and the tapetum degenerated. The pollen underwent mitosis to produce bicellular pollen; at which point, intine formation began, continuing through to pollen maturation. The entire cell wall development process was also punctuated by dynamic changes in pectin composition, particularly changes in methyl-esterified and de-methyl-esterified homogalacturonan.

Plant Protein O-Arabinosylation.

A wide range of proteins with diverse functions in development, defense, and stress responses are O-arabinosylated at hydroxyprolines (Hyps) within distinct amino acid motifs of continuous stretches of Hyps, as found in the structural cell wall extensins, or at non-continuous Hyps as, for example, found in small peptide hormones and a variety of plasma membrane proteins involved in signaling. Plant O-glycosylation relies on hydroxylation of Prolines to Hyps in the protein backbone, mediated by prolyl-4-hydroxylase (P4H) which is followed by O-glycosylation of the Hyp C4-OH group by either galactosyltransferases (GalTs) or arabinofuranosyltranferases (ArafTs) yielding either Hyp-galactosylation or Hyp-arabinosylation. A subset of the P4H enzymes with putative preference to hydroxylation of continuous prolines and presumably all ArafT enzymes needed for synthesis of the substituted arabinose chains of one to four arabinose units, have been identified and functionally characterized. Truncated root-hair phenotype is one common denominator of mutants of Hyp formation and Hyp-arabinosylation glycogenes, which act on diverse groups of O-glycosylated proteins, e.g., the small peptide hormones and cell wall extensins. Dissection of different substrate derived effects may not be regularly feasible and thus complicate translation from genotype to phenotype. Recently, lack of proper arabinosylation on arabinosylated proteins has been shown to influence their transport/fate in the secretory pathway, hinting to an additional layer of functionality of O-arabinosylation. Here, we provide an update on the prevalence and types of O-arabinosylated proteins and the enzymatic machinery responsible for their modifications.

Arabinogalactan glycoprotein dynamics during the progamic phase in the tomato pistil.

KEY MESSAGE: Pistil AGPs display dynamic localization patterns in response to fertilization in tomato. SlyFLA9 (Solyc07g065540.1) is a chimeric Fasciclin-like AGP with enriched expression in the ovary, suggesting a potential function during pollen-pistil interaction. During fertilization, the male gametes are delivered by pollen tubes to receptive ovules, deeply embedded in the sporophytic tissues of the pistil. Arabinogalactan glycoproteins (AGPs) are a diverse family of highly glycosylated, secreted proteins which have been widely implicated in plant reproduction, particularly within the pistil. Though tomato (Solanum lycopersicum) is an important crop requiring successful fertilization for production, the molecular basis of this event remains understudied. Here we explore the spatiotemporal localization of AGPs in the mature tomato pistil before and after fertilization. Using histological techniques to detect AGP sugar moieties, we found that accumulation of AGPs correlated with the maturation of the stigma and we identified an AGP subpopulation restricted to the micropyle that was no longer visible upon fertilization. To identify candidate pistil AGP genes, we used an RNA-sequencing approach to catalog gene expression in functionally distinct subsections of the mature tomato pistil (the stigma, apical and basal style and ovary) as well as pollen and pollen tubes. Of 161 predicted AGP and AGP-like proteins encoded in the tomato genome, we identified four genes with specifically enriched expression in reproductive tissues. We further validated expression of two of these, a Fasciclin-like AGP (SlyFLA9, Solyc07g065540.1) and a novel hybrid AGP (SlyHAE, Solyc09g075580.1). Using in situ hybridization, we also found SlyFLA9 was expressed in the integuments of the ovule and the pericarp. Additionally, differential expression analyses of the pistil transcriptome revealed previously unreported genes with enriched expression in each subsection of the mature pistil, setting the foundation for future functional studies.

Refractory alveolar rhabdomyosarcoma in an 11-year-old male.

Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% 5-yr disease-free survival rate when a chromosomal fusion is present and a 29% 5-yr disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion-negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-yr-old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.

Partial purification and immunodetection of cell surface glycoproteins from plants.

Cell surface glycoproteins in plants were first described more than 50 years ago, and yet, the precise mechanisms by which they operate remain elusive to this day. Studying glycoproteins is often challenging due to their subcellular localization (many secreted or membrane associated) and the extent of glycosylation present on the protein backbone, which can have profound effects on protein structure and behavior. In plants, additional layers of complexity exist as cell surface glycoproteins are in close contact, and in some cases, establish direct linkages with the polysaccharide networks present in the cell wall. In this chapter, we guide the reader through a protocol aimed to address the glycosylation status of a presumed cell surface glycoprotein. First, we discuss the advantages and disadvantages of using plants as homologous expression systems for recombinant glycoprotein production. Next, we describe a protocol for microsomal enrichment, followed by partial purification by affinity chromatography and finally glycodetection by immunoblotting using monoclonal antibodies targeting cell wall glycans. We particularly focus on the hydroxyproline-rich glycoprotein (HRGP) family, the most abundant family of glycoproteins in the plant cell wall. We provide examples of two putative HRGP chimeric proteins, one akin to extensins and the second an arabinogalactan protein (AGP)-like protein. For the latter, we provide an AGP-specific protocol to ensure enrichment of members of this group, which can be used independently or in conjunction with the described protocol. Throughout the chapter, we provide recommendations for the handling of plant glycoproteins and highlight special considerations for experimental design, along with troubleshooting suggestions.

Isolation and Cloning of Suppressor Mutants with Improved Pollen Fertility.

Mutant screens remain among the most powerful unbiased methods for identifying key genes in a pathway or process of interest. However, mutants impacting pollen function pose special challenges due to their genetic behavior. Here we describe an approach for isolating pollen mutants based on screening for suppressors of a low pollen fertility starting genotype. By identifying suppressor mutants with improved pollen fertility, we are able to identify new genes which are functionally relevant to pathway(s) causing low seed set in the original background. With this method, the low fertility of the genetic background may be due to one or more mutations or transgenes disrupting any aspect of pollen development or function. Furthermore, screening for improved pollen fertility biases toward recovery of the desired mutants due to their enhanced male transmission. The causative mutation is cloned using next-generation sequencing. The procedure uses both genetic and bioinformatics approaches to ultimately yield a very small list of candidate causative mutations speeding the transition from mutant phenotype to underlying gene.

Exocyst mutants suppress pollen tube growth and cell wall structural defects of hydroxyproline O-arabinosyltransferase mutants.

HYDROXYPROLINE O-ARABINOSYLTRANSFERASEs (HPATs) initiate a post-translational protein modification (Hyp-Ara) found abundantly on cell wall structural proteins. In Arabidopsis thaliana, HPAT1 and HPAT3 are redundantly required for full pollen fertility. In addition to the lack of Hyp-Ara in hpat1/3 pollen tubes (PTs), we also found broadly disrupted cell wall polymer distributions, particularly the conversion of the tip cell wall to a more shaft-like state. Mutant PTs were slow growing and prone to rupture and morphological irregularities. In a forward mutagenesis screen for suppressors of the hpat1/3 low seed-set phenotype, we identified a missense mutation in exo70a2, a predicted member of the vesicle-tethering exocyst complex. The suppressed pollen had increased fertility, fewer morphological defects and partially rescued cell wall organization. A transcriptional null allele of exo70a2 also suppressed the hpat1/3 fertility phenotype, as did mutants of core exocyst complex member sec15a, indicating that reduced exocyst function bypassed the PT requirement for Hyp-Ara. In a wild-type background, exo70a2 reduced male transmission efficiency, lowered pollen germination frequency and slowed PT elongation. EXO70A2 also localized to the PT tip plasma membrane, consistent with a role in exocyst-mediated secretion. To monitor the trafficking of Hyp-Ara modified proteins, we generated an HPAT-targeted fluorescent secretion reporter. Reporter secretion was partially dependent on EXO70A2 and was significantly increased in hpat1/3 PTs compared with the wild type, but was reduced in the suppressed exo70a2 hpat1/3 tubes.

Defining an embryonal rhabdomyosarcoma endotype.

Rhabdomyosarcoma (RMS) is the most common childhood soft-tissue sarcoma. The largest subtype of RMS is embryonal rhabdomyosarcoma (ERMS) and accounts for 53% of all RMS. ERMS typically occurs in the head and neck region, bladder, or reproductive organs and portends a promising prognosis when localized; however, when metastatic the 5-yr overall survival rate is ∼43%. The genomic landscape of ERMS demonstrates a range of putative driver mutations, and thus the recognition of the pathological mechanisms driving tumor maintenance should be critical for identifying effective targeted treatments at the level of the individual patients. Here, we report genomic, phenotypic, and bioinformatic analyses for a case of a 3-yr-old male who presented with bladder ERMS. Additionally, we use an unsupervised agglomerative clustering analysis of RNA and whole-exome sequencing data across ERMS and undifferentiated pleomorphic sarcoma (UPS) tumor samples to determine several major endotypes inferring potential targeted treatments for a spectrum of pediatric ERMS patient cases.