High-quality haplotype-resolved genome assembly for ring-cup oak (Quercus glauca) provides insight into oaks demographic dynamics.

Quercus section Cyclobalanopsis represents a dominant woody lineage in East Asian evergreen broadleaved forests. Regardless of its ecological and economic importance, little is known about the genomes of species in this unique oak lineage. Quercus glauca is one of the most widespread tree species in the section Cyclobalanopsis. In this study, a high-quality haplotype-resolved reference genome was assembled for Q. glauca from PacBio HiFi and Hi-C reads. The genome size, contig N50, and scaffold N50 measured 902.88, 7.60, and 69.28 Mb, respectively, for haplotype1, and 913.28, 7.20, and 71.53 Mb, respectively, for haplotype2. A total of 37,457 and 38,311 protein-coding genes were predicted in haplotype1 and haplotype2, respectively. Homologous chromosomes in the Q. glauca genome had excellent gene pair collinearity. The number of R-genes in Q. glauca was similar to most East Asian oaks but less than oak species from Europe and America. Abundant structural variation in the Q. glauca genome could contribute to environmental stress tolerance in Q. glauca. Sections Cyclobalanopsis and Cerris diverged in the Oligocene, in agreement with fossil records for section Cyclobalanopsis, which document its presence in East Asia since the early Miocene. The demographic dynamics of closely related oak species were largely similar. The high-quality reference genome provided here for the most widespread species in section Cyclobalanopsis will serve as an essential genomic resource for evolutionary studies of key oak lineages while also supporting studies of interspecific introgression, local adaptation, and speciation in oaks.

Plasma proteomic analysis may identify new markers for radiation-induced lung toxicity in patients with non-small-cell lung cancer.

PURPOSE: To study whether radiation induces differential changes in plasma proteomics in patients with and without radiation-induced lung toxicity (RILT) of Grade >/=2 (RILT2). METHODS AND MATERIALS: A total of 57 patients with NSCLC received radiation therapy (RT) were eligible. Twenty patients, 6 with RILT2 with tumor stage matched to 14 without RILT2, were enrolled for this analysis. Platelet-poor plasma was obtained before RT, at 2, 4, 6 weeks during RT, and 1 and 3 months after RT. Plasma proteomes were compared using a multiplexed quantitative proteomics approach involving ExacTag labeling, reverse-phase high-performance liquid chromatography and nano-LC electrospray tandem mass spectrometry. Variance components models were used to identify the differential protein expression between patients with and without RILT2. RESULTS: More than 100 proteins were identified and quantified. After excluding proteins for which there were not at least 2 subjects with data for at least two time points, 76 proteins remained for this preliminary analysis. C4b-binding protein alpha chain, Complement C3, and Vitronectin had significantly higher expression levels in patients with RILT2 compared with patients without RILT2, based on both the data sets of RT start to 3 months post-RT (p < 0.01) and RT start to the end of RT (p < 0.01). The expression ratios of patients with RILT2 vs. without RILT2 were 1.60, 1.36, 1.46, and 1.66, 1.34, 1.46, for the above three proteins, respectively. CONCLUSIONS: This proteomic approach identified new plasma protein markers for future studies on RILT prediction.

Radiation produces differential changes in cytokine profiles in radiation lung fibrosis sensitive and resistant mice.

BACKGROUND: Recent research has supported that a variety of cytokines play important roles during radiation-induced lung toxicity. The present study is designed to investigate the differences in early cytokine induction after radiation in sensitive (C57BL/6) and resistant mice (C3H). RESULTS: Twenty-two cytokines in the lung tissue homogenates, bronchial lavage (BAL) fluids, and serum from 3, 6, 12, 24 hrs to 1 week after 12 Gy whole lung irradiation were profiled using a microsphere-based multiplexed cytokine assay. The majority of cytokines had similar baseline levels in C57BL/6 and C3H mice, but differed significantly after radiation. Many, including granulocyte colony-stimulating factor (G-CSF), interleukin-6 (IL-6), and keratinocyte-derived chemokine (KC) were elevated significantly in specimens from both strains. They usually peaked at about 3-6 hrs in C57BL/6 and 6-12 hrs in C3H. At 6 hrs in lung tissue, G-CSF, IL-6, and KC increased 6, 8, and 11 fold in C57BL/6 mice, 4, 3, and 3 fold in the C3H mice, respectively. IL-6 was 10-fold higher at 6 hrs in the C57BL/6 BAL fluid than the C3H BAL fluid. MCP-1, IP-10, and IL-1alpha also showed some differences between strains in the lung tissue and/or serum. For the same cytokine and within the same strain of mice, there were significant linear correlations between lung tissue and BAL fluid levels (R2 ranged 0.46-0.99) and between serum and tissue (R2 ranged 0.56-0.98). CONCLUSION: Radiation induced earlier and greater temporal changes in multiple cytokines in the pulmonary fibrosis sensitive mice. Positive correlation between serum and tissue levels suggests that blood may be used as a surrogate marker for tissue.

Membrane glycoproteins associated with breast tumor cell progression identified by a lectin affinity approach.

The membrane glycoprotein component of the cellular proteome represents a promising source for potential disease biomarkers and therapeutic targets. Here we describe the development of a method that facilitates the analysis of membrane glycoproteins and apply it to the differential analysis of breast tumor cells with distinct malignant phenotypes. The approach combines two membrane extraction procedures, and enrichment using ConA and WGA lectin affinity columns, prior to digestion and analysis by LC-MS/MS. The glycoproteins are identified and quantified by spectral counting. Although the distribution of glycoprotein expression as a function of MW and p I was very similar between the two related cell lines tested, the approach enabled the identification of several distinct membrane glycoproteins with an expression index correlated with either a precancerous (MCF10AT1), or a malignant, metastatic cellular phenotype (MCF10CA1a). Among the proteins associated with the malignant phenotype, Gamma-glutamyl hydrolase, CD44, Galectin-3-binding protein, and Syndecan-1 protein have been reported as potential biomarkers of breast cancer.

The use of blood biomarkers to predict radiation lung toxicity: a potential strategy to individualize thoracic radiation therapy.

BACKGROUND: Radiation-induced lung toxicity (RILT) is an important dose-limiting toxicity during thoracic radiotherapy. Early prediction of radiation lung toxicity will allow physicians to determine a customized treatment regimen for each patient and deliver a radiation dose tailored to that individual's normal tissue sensitivity profile rather than to the average tolerance of the whole population. METHODS: This review focuses on blood biomarkers in predicting radiation-induced lung toxicity. We searched the literature for data associated with cytokines, and we review the updates of proteomic and genetic polymorphisms in radiation lung toxicity. RESULTS: Studies from single institutions have demonstrated the significant values of cytokines such as TGF-beta1, IL-6, KL-6, surfactant proteins, and IL-1ra on predicting RILT. The majority of studies focus on the values prior to and at the end of radiation therapy. There is limited data from proteomics and specific genomic single nucleotide polymorphism studies that target individualized radiation therapy for patients with lung cancer. CONCLUSIONS: Biomarkers or models that can accurately predict radiation-induced lung damage at an early stage, before completion of chemoradiation, would allow physicians to monitor and customize remaining treatment for each patient.

Comparative proteomic analysis of radiation-induced changes in mouse lung: fibrosis-sensitive and -resistant strains.

To determine whether comparative proteomics could detect differential protein expression after lung irradiation in two mouse strains with different radiation responses, lung proteins were subjected to two-dimensional orthogonal liquid-phase separations, with chromatofocusing in the first dimension and nonporous silica reverse-phase high-performance liquid chromatography (NPS-RP-HPLC) in the second. Five weeks after 12 Gy whole-lung irradiation, 15 and 31 proteins had significantly altered expression levels in C3H/HeJ (less likely to develop lung fibrosis) and C57BL/6J mice (more likely to develop lung fibrosis), respectively. These proteins were analyzed by HPLC-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) and identified by matching sequences in a peptide database. The proteins are associated with redox, energy consumption, glycolysis, or chromatin/ RNA structure formation. Five of the six redox-related proteins, including superoxide dismutase 1 (SOD1), cytochrome c oxidase, glutamate dehydrogenase, biliverdin reductase, peroxiredoxin and carbonyl reductase, were down-regulated in the irradiated C57BL/6J mice, whereas SOD1, sulfurtransferase and carbonyl reductase increased in the irradiated C3H/ HeJ mice. Thus decreased antioxidant proteins in the irradiated C57BL/6J mice may be correlated with increased early lung toxicity. Changes in SOD1 and 8-hydroxydeoxy-guanosine (8-OHdG, an oxidative stress marker) were further confirmed by immunohistochemistry and/or Western blot analysis. These data suggest that a proteomics approach has the potential to detect protein changes relevant to early lung toxicity after irradiation.

Microdialysis sampling of cytokines.

Microdialysis sampling is a well-known method for collection of low molecular weight hydrophilic analytes. Due to the success of this sampling technique for these analytes, many researchers have wanted to extend the use of this method to a wider range of analytes-particularly proteins and peptides. These analytes pose unique challenges during microdialysis sampling. The primary challenges are the reduced recovery across the dialysis semi-permeable membrane and the volume limitations/requirements for the typical immunoassay methods used for detection of proteins. This review covers the practical and theoretical aspects needed for in vivo microdialysis sampling of cytokines, which are a vitally important class of signaling proteins. In addition to the basics of the microdialysis method for sampling cytokines, the use of the microdialysis device as a localized cytokine delivery method is also described. Since relative recovery of cytokines is often low during microdialysis sampling, methods to improve the membrane recovery are discussed for in vitro and in vivo applications.

No delayed temporal response to sample concentration changes during enhanced microdialysis sampling using cyclodextrins and antibody-immobilized microspheres.

The temporal response to concentration changes external to a microdialysis probe containing trapping agents in the perfusion fluid was studied. Native beta-cyclodextrin and a water-soluble beta-cyclodextrin polymer were used as trapping agents in the microdialysis perfusion fluid to study the temporal concentration response to carbamazepine, a hydrophobic analyte. The temporal response of microdialysis probes containing antibody-immobilized microspheres against five different cytokines (tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-5) to concentration changes outside of the probe was also determined. In both cases, no delayed temporal response of enhanced microdialysis was observed for either carbamazepine or the cytokines as compared to standard microdialysis sampling procedures.

Multiplexed cytokine detection in microliter microdialysis samples obtained from activated cultured macrophages.

Microdialysis sampling probes were used to collect cytokine samples from lipopolysaccharide (LPS)-stimulated macrophages. The probes were immersed into cell culture wells containing either RAW 264.7 or isolated peritoneal macrophages. Dialysates (15 microL) from these wells were subjected to a multiplexed cytokine sandwich immunoassay platform analyzed by flow cytometry that measures up to six separate cytokines, interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12p70 (IL-12p70), interferon-gamma (IFN-gamma), macrophage chemoattractant protein-1 (MCP-1), and tumor necrosis factor-alpha (TNF-alpha) in a single 15-muL sample. In vitro microdialysis sampling relative recovery experiments showed that only IFN-gamma, IL-6, MCP-1, and TNF-alpha could be recovered across a commercially-available 100-kDa MWCO microdialysis membrane. Eleven hours after LPS addition (1 microg/mL), RAW 264.7 macrophages secreted greater than 8000 pg/mL of TNF-alpha and greater than 1000 pg/mL MCP-1. With the peritoneal macrophages, greater than 6000 pg/mL of IL-6, MCP-1, and TNF-alpha were obtained. The maximum dialysate concentrations obtained from the RAW macrophages were 1300 pg/mL for TNF-alpha and 55 pg/mL for MCP-1. Maximum cytokine concentrations from peritoneal macrophage dialysates reached approximately 2000 pg/mL, 1100 pg/mL and 500 pg/mL for TNF-alpha, MCP-1 and IL-6, respectively. Microdialysis sampling allowed for 20-min samples to be collected during the cytokine release from the activated macrophages. These results demonstrate that microdialysis sampling can be used for collection of selected cytokines with improved temporal resolution.

In vivo microdialysis sampling of cytokines produced in mice given bacterial lipopolysaccharide.

Cytokines are proteins that mediate communication between cells of the immune system as well as certain other non-immune host cells. These proteins are produced by many cell types and they mediate immune and inflammatory responses. However, the direct site analysis of these critical proteins is hampered by the lack of site-specific tools available for such direct measurements. In this study, both in vitro and in vivo microdialysis sampling of different cytokines (tumor necrosis factor-alpha [TNF-alpha], interferon-gamma [IFN-gamma], interleukin-6 [IL-6], IL-12p70, and macrophage chemoattractant protein-1 [MCP-1]) was performed. A mouse model of bacterial lipopolysaccharide (LPS) administration and response pattern was used for in vivo studies. Three cytokines, TNF-alpha, IL-6, and MCP-1 were quantified in the serum from mice given LPS. In vivo studies demonstrated the ability to monitor increasing levels of these cytokines (TNF-alpha, IL-6, and MCP-1) via microdialysis probes placed in the peritoneal cavity of mice given LPS. All three cytokines were quantified simultaneously in 15 muL of dialysate using a multiplexed bead-based immunoassay for flow cytometry. The detected dialysate cytokine concentrations varied between 200 pg/mL and 1500 pg/mL for TNF-alpha, between 600 pg/mL and 3000 pg/mL for MCP-1, and between 2700 pg/mL and more than 5000 pg/mL for IL-6. The detected serum cytokine concentrations ranged from 5700 pg/mL to 35,000 pg/mL for TNF-alpha, from 40,000 pg/mL to 65,000 pg/mL for MCP-1, and greater than than 100,000 pg/mL for IL-6. This work demonstrates that microdialysis sampling can be used in vivo to collect temporal profiles of cytokine production.

Enhanced microdialysis relative recovery of inflammatory cytokines using antibody-coated microspheres analyzed by flow cytometry.

Achieving high relative recovery (RR) of proteins during microdialysis sampling is difficult due to the diffusion limitations inherent to this sampling process. This often causes low microdialysis RR for proteins with molecular weight >10 kDa. A RR enhancement process for microdialysis sampling of proteins has been developed that can be readily used with flow cytometers. Multiplexed RR enhancement and detection of five different cytokines (TNF-alpha, IFN-gamma, IL-2, IL-4, and IL-5) was achieved by including antibody-coated microspheres in the microdialysis perfusion fluid. Inclusion of these antibody-coated microspheres causes an increase in the analyte diffusive driving force across the dialysis membrane and a subsequent increase in the relative recovery. For the five cytokines, typical control and enhanced relative recoveries at a 1.0 microL/min flow rate were as follows (n = 3): TNF-alpha, 5.5 +/- 0.6% and 60.4 +/- 5.8%; IFN-gamma, 2.6 +/- 0.3% and 25.8 +/- 2.3%; IL-5, 1.4 +/- 0.2% and 4.9 +/- 0.1%; IL-4, 10.9 +/- 0.6% and 78.8 +/- 8.0%; and IL-2, 4.1 +/- 0.4% and 19.8 +/- 2.5%. Using this approach, a four- to 12-fold enhancement of microdialysis RR was achieved for the five cytokines from a quiescent solution. The enhancement varies among the five cytokines and may be due to different diffusive and antibody binding properties. TNF-alpha exhibited the highest RR enhancement, while IL-5 exhibited the lowest. Experimental parameters that affect the enhancement, such as flow rate, sample collection volume, and bead density, were studied.

Water-soluble cyclodextrin polymers for enhanced relative recovery of hydrophobic analytes during microdialysis sampling.

Microdialysis relative recovery (RR) enhancement using different water-soluble, epichlorohydrin-based cyclodextrin polymers (CD-EPS) was studied in vitro for different analytes, amitryptiline, carbamazepine, hydroquinone, ibuprofen, and 4-nitrophenol. When compared to the native CDs (alpha, beta, and gamma) on a per mole basis, the CD-EPS enhanced microdialysis RR was either statistically greater or the same. beta-CD-EPS was more highly retained than native beta-CD by a 20 000 Da molecular weight cutoff (MWCO) polycarbonate membrane, but showed no statistical difference for loss across a 100 000 Da MWCO polyethersulfone membrane (PES). When the same weight percent of beta-CD or beta-CD-EPS was included in the microdialysis perfusion fluid, the beta-CD-EPS produced a higher microdialysis RR than native beta-CD for all analytes across the PES membrane. However, enhancements for the PC membrane were statistically insignificant when beta-CD and beta-CD-EPS were compared on a per mole basis. These results suggest that CD-EPS may be used as effective enhancement agents during microdialysis sampling and for some membranes provide the additional advantage of being retained more than native CDs.