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The peripheral immune system response to Intracerebral Hemorrhage (ICH) may differ with ICH in different brain locations. Thus, we investigated peripheral blood mRNA expression of Deep ICH, Lobar ICH, and vascular risk factor-matched control subjects (n = 59). Deep ICH subjects usually had hypertension. Some Lobar ICH subjects had cerebral amyloid angiopathy (CAA). Genes and gene networks in Deep ICH and Lobar ICH were compared to controls. We found 774 differentially expressed genes (DEGs) and 2 co-expressed gene modules associated with Deep ICH, and 441 DEGs and 5 modules associated with Lobar ICH. Pathway enrichment showed some common immune/inflammatory responses between locations including Autophagy, T Cell Receptor, Inflammasome, and Neuroinflammation Signaling. Th2, Interferon, GP6, and BEX2 Signaling were unique to Deep ICH. Necroptosis Signaling, Protein Ubiquitination, Amyloid Processing, and various RNA Processing terms were unique to Lobar ICH. Finding amyloid processing pathways in blood of Lobar ICH patients suggests peripheral immune cells may participate in processes leading to perivascular/vascular amyloid in CAA vessels and/or are involved in its removal. This study identifies distinct peripheral blood transcriptome architectures in Deep and Lobar ICH, emphasizes the need for considering location in ICH studies/clinical trials, and presents potential location-specific treatment targets.

Renal oncocytoma (RO) accounts for 5% of renal cancers and generally behaves as a benign tumor with favorable long-term prognosis. It is difficult to confidently distinguish between benign RO and other renal malignancies, particularly chromophobe renal cell carcinoma (chRCC). Therefore, RO is often managed aggressively with surgery. We sought to identify molecular biomarkers to distinguish RO from chRCC and other malignant renal cancer mimics. In a 44-patient discovery cohort, we identified a significant differential abundance of nine genes in RO relative to chRCC. These genes were used to train a classifier to distinguish RO from chRCC in an independent 57-patient cohort. The trained classifier was then validated in five independent cohorts comprising 89 total patients. This nine-gene classifier trained on the basis of differential gene expression showed 93% sensitivity and 98% specificity for distinguishing RO from chRCC across the pooled validation cohorts, with a c-statistic of 0.978. This tool may be a useful adjunct to other diagnostic modalities to decrease the diagnostic and management uncertainty associated with small renal masses and to enable clinicians to recommend more confidently less aggressive management for some tumors. PATIENT SUMMARY: Renal oncocytoma is generally a benign form of kidney cancer that does not necessarily require surgical removal. However, it is difficult to distinguish renal oncocytoma from other more aggressive forms of kidney cancer, so it is treated most commonly with surgery. We built a classification tool based on the RNA levels of nine genes that may help avoid these surgeries by reliably distinguishing renal oncocytoma from other forms of kidney cancer.

Fast-developing single-cell multimodal omics (scMulti-omics) technologies enable the measurement of multiple modalities, such as DNA methylation, chromatin accessibility, RNA expression, protein abundance, gene perturbation, and spatial information, from the same cell. scMulti-omics can comprehensively explore and identify cell characteristics, while also presenting challenges to the development of computational methods and tools for integrative analyses. Here, we review these integrative methods and summarize the existing tools for studying a variety of scMulti-omics data. The various functionalities and practical challenges in using the available tools in the public domain are explored through several case studies. Finally, we identify remaining challenges and future trends in scMulti-omics modeling and analyses.

RNA expression of endogenous retroviral elements poses a threat to the genome and to cell function, which may ultimately result in disease. Recently published in Nature, Chelmicki and colleagues (2021) identify m6A mRNA methylation as a form of regulation to defend the cell against these attacks.

The epic arms race between microbes and their predators was the driving force behind the evolution and diversification of the truly remarkable microbial adaptive immune system CRISPR-Cas. CRISPR-Cas systems mediate defense through three stages: recording of nucleic acid species, multiplexed RNA expression and processing, and eventually RNA-guided cleavage of hostile genetic elements. The entire process is orchestrated by a plethora of effector proteins endowed with specialized functions for manipulating genetic material. Investigating this treasure trove substantially fostered the development of the RNA-guided DNA endonuclease Cas9 into a versatile molecular tool for synthetic biology and biomedicine. Here, we review the developments of Cas9 and other CRISPR-Cas components for applications in synthetic biology as well as highlight emerging CRISPR-Cas-based genetic recorders and memory devices.

Neuronal membrane potential is of fundamental importance for the mechanistic understanding of brain function. This review discusses progress in whole-cell patch-clamp recordings for low-noise measurement of neuronal membrane potential in awake behaving animals. Whole-cell recordings can be combined with two-photon microscopy to target fluorescently labeled neurons, revealing cell-type-specific membrane potential dynamics of retrogradely or genetically labeled neurons. Dual whole-cell recordings reveal behavioral modulation of membrane potential synchrony and properties of synaptic transmission in vivo. Optogenetic manipulations are also readily integrated with whole-cell recordings, providing detailed information about the effect of specific perturbations on the membrane potential of diverse types of neurons. Exciting developments for future behavioral experiments include dendritic whole-cell recordings and imaging, and use of the whole-cell recording pipette for single-cell delivery of drugs and DNA, as well as RNA expression profiling. Whole-cell recordings therefore offer unique opportunities for investigating the neuronal circuits and synaptic mechanisms driving membrane potential dynamics during behavior.

We have carried an exploratory study by blood transcriptome to find RNA expression signatures in familial ADHD. Samples were collected from three cases with familial ADHD and their paired controls and evaluated by RNA-Seq. Transcriptome profiling identified 7 differentially expressed transcripts with a FDR0.05 that were involved in pathways in Huntington's disease or axonal guidance signaling previously implicated in ADHD, and enriched for signal peptide, growth factor binding, and notably the lipid metabolism pathways. These findings show that blood transcriptome can have an associated signature and highlight a potential to use blood transcriptome to identify patterns of ADHD.

The most popular CRISPR-SpCas9 system recognizes canonical NGG protospacer adjacent motifs (PAMs). Previously engineered SpCas9 variants, such as Cas9-NG, favor G-rich PAMs in genome editing. In this manuscript, we describe a new plant genome-editing system based on a hybrid iSpyMacCas9 platform that allows for targeted mutagenesis, C to T base editing, and A to G base editing at A-rich PAMs. This study fills a major technology gap in the CRISPR-Cas9 system for editing NAAR PAMs in plants, which greatly expands the targeting scope of CRISPR-Cas9. Finally, our vector systems are fully compatible with Gateway cloning and will work with all existing single-guide RNA expression systems, facilitating easy adoption of the systems by others. We anticipate that more tools, such as prime editing, homology-directed repair, CRISPR interference, and CRISPR activation, will be further developed based on our promising iSpyMacCas9 platform., This study reports the development of an iSpyMacCas9 genome-editing toolbox for targeted mutagenesis, C to T base editing, and A to G base editing at A-rich PAM sites. While the tools are demonstrated in rice, they are expected to aid genome-editing applications in other plant species as well with a broadened targeting scope.

Objective The present study was carried out to unfold the clinical significance of apoptosis stimulating protein of p53 (ASPP) 1 and ASPP2 expression in oral cancer (OC). Methods Tissue specimens (malignant and their corresponding adjacent normal) from 40 pathologically confirmed OC patients treated at the Institute were included in the study. ASPP1 and ASPP2 expression were examined using semi-quantitative RT-PCR. Results The results indicated lower ASPP1 expression in OC tissues as compared to adjacent normal tissues (p = 0.085). Stratified analysis as per tumor site revealed significant down-regulation of ASPP1 in tongue cancer tissues (p = 0.005). Receiver operating characteristic curve depicted significant discriminatory efficacy in distinguishing tongue cancer tissues and adjacent normal tissues (p = 0.019). Moreover, ASPP1 expression was remarkably declined in stage II, III and IV OC tumors than stage I OC tumors (p = 0.007, 0.092 and 0.013, respectively). A similar trend was observed in buccal mucosa tumors on further analysis. ASPP2 expression was lower in moderately differentiated OC tumors as compared to well differentiated OC tumors (p = 0.061). Significantly reduced ASPP2 expression was observed in tongue cancer tumors without invasion in contrast to tumors with perineural invasion (p = 0.007). Besides, ASPP1 and ASPP2 expression was positively inter-correlated in tongue tissues (r = 0.325, p = 0.091). Conclusions Lower ASPP1 expression in tongue cancer during malignant transformation has significance in cancer initiation. Association of reduced ASPP1 and ASPP2 expression with advanced disease stage and moderate differentiation suggests their role in OC progression. Thus, down-regulation of ASPP1 and ASPP2 may serve as potential diagnostic and prognostic indicators in OC.

In forensic genetics, the RNA expression profiles obtained from forensic casework can be utilized to identify specimens’ biological features, such as the origin of body fluids, age estimation, etc. However, RNA is grossly susceptible to external factors and this characteristic leads to its low integrity and concentration, which make the ideal RNA expression profiles hard to be obtained. Thus, the methods of sample pretreatment and storage play a pivotal role in forensic RNA researches. In this study, we evaluated three different pretreatment methods for their effects on the quality of RNA extracted from peripheral blood by detecting the RNA yield, concentration and integrity. Results illustrated that the RNA pretreated by the preservation solution and stored at −80 °C had the highest quality.

Understanding individual susceptibility to drug-induced cardiotoxicity is key to improving patient safety and preventing drug attrition. Human induced pluripotent stem cells (hiPSCs) enable the study of pharmacological and toxicological responses in patient-specific cardiomyocytes (CMs), and may serve as preclinical platforms for precision medicine. Transcriptome profiling in hiPSC-CMs from seven individuals lacking known cardiovascular disease-associated mutations, and in three isogenic human heart tissue and hiPSC-CM pairs, showed greater inter-patient variation than intra-patient variation, verifying that reprogramming and differentiation preserve patient-specific gene expression, particularly in metabolic and stress-response genes. Transcriptome-based toxicology analysis predicted and risk-stratified patient-specific susceptibility to cardiotoxicity, and functional assays in hiPSC-CMs using tacrolimus and rosiglitazone, drugs targeting pathways predicted to produce cardiotoxicity, validated inter-patient differential responses. CRISPR/Cas9-mediated pathway correction prevented drug-induced cardiotoxicity. Our data suggest that hiPSC-CMs can be used in vitro to predict and validate patient-specific drug safety and efficacy, potentially enabling future clinical approaches to precision medicine.

Hippocampus-dependent learning is known to induce changes in gene expression, but information on gene expression differences between different learning paradigms that require the hippocampus is limited. The bulk of studies investigating RNA expression after learning use the contextual fear conditioning task, which couples a novel environment with a footshock. Although contextual fear conditioning has been useful in discovering gene targets, gene expression after spatial memory tasks has received less attention. In this study, we used the object-location memory task and studied gene expression at two time points after learning in a high-throughput manner using a microfluidic qPCR approach. We found that expression of the classic immediate-early genes changes after object-location training in a fashion similar to that observed after contextual fear conditioning. However, the temporal dynamics of gene expression are different between the two tasks, with object-location memory producing gene expression changes that last at least 2 hours. Our findings indicate that different training paradigms may give rise to distinct temporal dynamics of gene expression after learning.

Many RNAs present unique challenges in obtaining material suitable for structural or biophysical characterization. These issues include synthesis of chemically and conformationally homogeneous RNAs, refolding RNA purified using denaturing preparation techniques, and avoiding chemical damage. To address these challenges, new methodologies in RNA expression and purification have been developed seeking to emulate those commonly used for protein purifications. In this review, recent developments in the preparation of high-quality RNA for structural biology and biophysical applications are discussed, with an emphasis on native methods.

Background T + chemo is standard 1st line therapy for HER2+ gastroesophageal adenocarcinoma; however, patients (pts) tend to progress in 6-8 months. Up to 40% show loss of HER2 expression post T, likely underlying the lack of efficacy of anti-HER2 agents in 2nd line therapy. We report here a clinical update and biomarker analysis of an ongoing study in pts receiving M, an anti-HER2 Fc-optimized mAb, plus P in HER2+ GEA pts in post T, 2nd line, chemotherapy-free treatment. Methods Endpoints described herein are safety, objective response rate (ORR), disease control rate (DCR), archival HER2 IHC level and/or ERBB2 amplification (amp) status pre-M+P in cell free DNA (cfDNA) by NGS (Guardant360), PD-L1 CPS by IHC (22C3 pharmDx), anti-HER2 T- cell immunity by ELISPOT on PBMCs, and NanoString PanCancer IO360™ assay on archival FFPE biopsies. Results 92 pts (66% GC, 34% GEJ) received 15mg/kg M+200mg P Q3W. 99% were MSS and 43% were PD-L1+(>1%). Related adverse events ≥grade 3 were 18.5%. Confirmed ORR was 19% with 54% DCR; interestingly, higher response rates occurred in GC vs GEJ (25% vs 7% ORR, p=0.047 and 64% vs 33% DCR, p=0.008). A higher proportion of GC vs GEJ cancers was HER2 3+ on archival IHC (90% vs 53%, p=0.0075), furthermore, a 5.3-fold greater fraction of GC vs GEJ pts showed upregulated tumor ERBB2 RNA expression (p 6-fold higher in GC vs GEJ (34.7% vs 5.5%, p=0.03) and associated with 53% confirmed ORR and 82% DCR in GC. GC pts with higher baseline T-cell immunity toward HER2 (p59 class II peptide) had greater probability of ORR (p=0.005). Conclusions In this study, M+P demonstrated tolerability and preliminary evidence of anti-tumor activity as a chemotherapy-free regimen in 2nd line HER2+ GC; biomarker analysis suggests an association with higher expression and retention of HER2 and PD-L1 together with pre-existing anti-HER2 immunity. Clinical trial identification NCT02689284. Legal entity responsible for the study MacroGenics, Inc. Funding MacroGenics, Inc. Disclosure H. Park: Research grant / Funding (institution): Ambrx; Research grant / Funding (institution): Amgen; Research grant / Funding (institution): Array BioPharma; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Bayer; Research grant / Funding (institution): BeiGene; Research grant / Funding (institution): Bristol-Myers Squibb; Research grant / Funding (institution): Daiichi Sankyo; Research grant / Funding (institution): EMD Serono; Research grant / Funding (institution): Gilead Sciences; Research grant / Funding (institution): GlaxoSmithKline; Research grant / Funding (institution): Incyte; Research grant / Funding (institution): Lilly; Research grant / Funding (institution): MacroGenics; Research grant / Funding (institution): MedImmune; Research grant / Funding (institution): Medivation; Research grant / Funding (institution): Merck; Research grant / Funding (institution): Millennium; Research grant / Funding (institution): Novartis; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Puma Biotechnology; Research grant / Funding (institution): Regeneron; Research grant / Funding (institution): Roche; Research grant / Funding (institution): Taiho Pharmaceutical; Research grant / Funding (institution): Vertex. H. Uronis: Shareholder / Stockholder / Stock options, Full / Part-time employment: GeneCentric; Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Bristol-Myers Squibb; Research grant / Funding (institution): Advaxis; Research grant / Funding (institution): Genentech/Roche; Research grant / Funding (institution): Lycera; Research grant / Funding (institution): MacroGenics; Research grant / Funding (institution): Merck. Y. Kang: Advisory / Consultancy: Bristol-Myers Squibb; Advisory / Consultancy, Research grant / Funding (institution): DAE HWA Pharmaceutical; Advisory / Consultancy: Lilly/Imclone; Advisory / Consultancy: Merck/Serano; Advisory / Consultancy: Ono Pharmaceutical; Advisory / Consultancy: Roche/Genentech; Research grant / Funding (institution): Taiho Pharmaceutical; Research grant / Funding (institution): LSK Biopharma. M.C.H. Ng: Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: MSD Oncology; Honoraria (self), Travel / Accommodation / Expenses: Taiho Pharmaceutical; Advisory / Consultancy: Bristol-Myers Squibb; Research grant / Funding (institution): ASLAN Pharmaceuticals. P. Enzinger: Advisory / Consultancy: Astellas Pharma; Advisory / Consultancy: Five Prime Therapeutics; Advisory / Consultancy: Merck; Advisory / Consultancy: Taiho Pharmaceutical. K.W. Lee: Research grant / Funding (institution): Array BioPharma; Research grant / Funding (institution): ASLAN Pharmaceuticals; Research grant / Funding (institution): AstraZeneca/MedImmune; Research grant / Funding (institution): Five Prime Therapeutics; Research grant / Funding (institution): Green Cross Corp; Research grant / Funding (institution): LSK BioPharma; Research grant / Funding (institution): MacroGenics; Research grant / Funding (institution): Merck KGaA; Research grant / Funding (institution): MSD; Research grant / Funding (institution): Ono Pharmaceutical; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Pharmacyclics; Honoraria (self): Bristol-Myers Squibb; Honoraria (self): Lilly; Research grant / Funding (institution): ALX Oncology. S. Rutella: Leadership role: Clinical & Biomarkers Data Sharing Subcommittee, Society for Immunotherapy of Cancer; Research grant / Funding (institution): John and Lucille van Geest Foundation. S.E. Church: Shareholder / Stockholder / Stock options, Full / Part-time employment: NanoString Technologies. J. Nordstrom: Shareholder / Stockholder / Stock options, Full / Part-time employment: MacroGenics, Inc. T. Wu: Shareholder / Stockholder / Stock options, Full / Part-time employment: MacroGenics, Inc. J. Yen: Shareholder / Stockholder / Stock options, Full / Part-time employment: Guardant Health. A. Franovic: Shareholder / Stockholder / Stock options, Full / Part-time employment: Guardant Health. J. Muth: Shareholder / Stockholder / Stock options, Full / Part-time employment: MacroGenics, Inc. M. Rosales: Shareholder / Stockholder / Stock options, Full / Part-time employment: MacroGenics, Inc. J. Davidson-Moncada: Shareholder / Stockholder / Stock options, Full / Part-time employment: MacroGenics, Inc. Y. Bang: Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca/MedImmune; Advisory / Consultancy, Research grant / Funding (institution): Bayer; Advisory / Consultancy, Research grant / Funding (institution): Beigene; Advisory / Consultancy, Research grant / Funding (institution): Green Cross; Advisory / Consultancy: Hanmi; Advisory / Consultancy, Research grant / Funding (institution): Merck Serono; Advisory / Consultancy, Research grant / Funding (institution): MSD Oncology; Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy: Samyang; Advisory / Consultancy, Research grant / Funding (institution): Taiho Pharmaceutical; Research grant / Funding (institution): Astellas Pharma; Research grant / Funding (institution): Boehringer Ingelheim; Research grant / Funding (institution): Boston Biomedical; Research grant / Funding (institution): Bristol-Myers Squibb; Research grant / Funding (institution): CKD; Research grant / Funding (institution): Curis; Research grant / Funding (institution): Daiichi Sankyo; Research grant / Funding (institution): Five Prime Therapeutics; Research grant / Funding (institution): Genentech/Roche; Research grant / Funding (institution): GlaxoSmithKline; Research grant / Funding (institution): Lilly; Research grant / Funding (institution): MacroGenics; Research grant / Funding (institution): Ono Pharmaceutical; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Takeda. D.V. Catenacci: Honoraria (self), Advisory / Consultancy: Amgen; Honoraria (self), Advisory / Consultancy: Bristol-Myers Squibb; Honoraria (self): Five Prime Therapeutics; Honoraria (self), Speaker Bureau / Expert testimony: Foundation Medicine; Honoraria (self), Advisory / Consultancy: Genentech/Roche; Honoraria (self): GenMab; Honoraria (self): Gritstone Oncology; Honoraria (self), Speaker Bureau / Expert testimony: Guardant Health; Honoraria (self), Advisory / Consultancy: Lilly; Honoraria (self): Merck; Honoraria (self): NantOmics; Honoraria (self): OncoPlex Diagnostics; Honoraria (self), Advisory / Consultancy: Taiho Pharmaceutical; Advisory / Consultancy: Astellas Pharma. All other authors have declared no conflicts of interest.

Little is known about the role of adipokines in the pathogenesis of coronary artery disease in young patients. The aims of this study were to compare serum levels of adipokines and expression of adipokines in peripheral blood leukocytes in patients with premature coronary artery disease (CAD), metabolic syndrome and healthy individuals.Sixty-five patients with premature CAD (men 18-45years old and women 18-55years old) formed the study group. The control groups were 75 patients with metabolic syndrome and 50 healthy individuals. For each group, RNA expression in peripheral blood leukocytes was determined for 24 different adipokines and 11 adipokines were examined in serum.In individuals with CAD, serum visfatin levels were significantly higher than in metabolic syndrome and healthy controls (2.3 vs. 1.6 vs. 0.7µg/L, P0.001) while both omentin-1 (92.9 vs. 587.0 vs. 552.3µg/L, P0.001) and ZAG2 (45.5 vs. 72.5 vs. 77.1mg/L, P0.001) levels were lower. The receiver operating curve (ROC) analysis for testing the validity of these adipokines in the diagnosis of CAD compared to control groups provided the following areas under the curve (AUC): omentin-1 AUC 0.97 (cut-off ≤222µg/L), ZAG2 AUC 0.89 (cut-off ≤51.7mg/L) and visfatin AUC 0.74 (cut-off ≥1.0µg/L) (P0.001 in all cases). Visfatin and omentin-1 serum levels did not differ between the acute phase of myocardial infarction and the chronic phase of CAD. In patients with CAD, we found no significant relation between mRNA expression and adipokine concentration.Serum omentin-1, visfatin and ZAG2 could serve as biomarkers of premature CAD in young apparently healthy people.

RNA expression profiles are increasingly used to diagnose and classify disease, based on expression patterns of as many as several thousand RNAs. To ensure quality of expression profiling services in clinical settings, a standard operating procedure incorporates multiple quality indicators and controls, beginning with preanalytic specimen preparation and proceeding thorough analysis, interpretation, and reporting. Before testing, histopathological examination of each cellular specimen, along with optional cell enrichment procedures, ensures adequacy of the input tissue. Other tactics include endogenous controls to evaluate adequacy of RNA and exogenous or spiked controls to evaluate run- and patient-specific performance of the test system, respectively. Unique aspects of quality assurance for array-based tests include controls for the pertinent outcome signatures that often supersede controls for each individual analyte, built-in redundancy for critical analytes or biochemical pathways, and software-supported scrutiny of abundant data by a laboratory physician who interprets the findings in a manner facilitating appropriate medical intervention. Access to high-quality reagents, instruments, and software from commercial sources promotes standardization and adoption in clinical settings, once an assay is vetted in validation studies as being analytically sound and clinically useful. Careful attention to the well-honed principles of laboratory medicine, along with guidance from government and professional groups on strategies to preserve RNA and manage large data sets, promotes clinical-grade assay performance.

Background Atherosclerotic lesions predominantly localize in areas exposed to distinct hemodynamic conditions. In such lesions, tissue factor (TF) is over-expressed. Therefore, we hypothesized that varying types of mechanical forces may induce different effects on TF expression in endothelial cell, and may also influence the effects of chemical stimuli. Materials and Methods TF RNA expression in human umbilical vein endothelial cells (HUVEC) exposed to mechanical stress in the presence or absence of chemical stimulation with thrombin (Th) was determined. The forces examined were: steady unidirectional laminar flow (LF), pulsatile unidirectional laminar flow (PF), constant oscillatory flow (OF), pulsatile to-fro flow (TFF), and cyclic strain (CS). Results Mechanical stimulation of HUVEC with LF for 2 h induced an 8.7 ± 0.7-fold increase in TF RNA expression, while PF induced 4.7 ± 0.9 and TFF induced 8.6 ± 1.7-fold, respectively. These responses were significantly higher than static controls. Exposure to OF or CS did not result in any significant increase, whereas chemical stimulation with Th led to significant TF expression (4.9 ± 0.3-fold). The combination of mechanical-chemical stimuli induced significantly higher TF expression than mechanical stresses alone, and this effect was synergistic. Combination of LF+Th for 2 h induced significantly increased TF expression (16.6 ± 1.7-fold), as did PF+Th (14.8 ± 2.4) and TFF+Th (17.4 ± 1.0). Furthermore, after 6 h exposure, only TFF demonstrated significantly higher TF expression both with and without Th. Conclusions While uniform laminar flow resulted in transient TF expression, disturbed flow induced sustained amplification of TF expression. Further investigation is needed to elucidate the mechanism of localized atherosclerosis in areas exposed to disturbed flow.

The Hina gene is one of the two known Hin genes for hardness, and its RNA expression is correlated with grain hardness and dry matter digestibility variation. In this study, only one clone of Hina gene was obtained from one barley accession. A total of 121 Hina gene sequences were isolated from 121 wild barley (Hordeum spontaneum) accessions in Israel, Iran, and Turkey, and then their molecular characteristics were compared with 97 Hina gene sequences from 74 cultivated barley (H. vulgare) lines in Europe and 23 landrace (H. vulgare) with global distribution and other 26 Hina gene sequences from cultivated barleys (H. vulgare) with unknown global distribution. Cis-acting regulatory element (CARE) searching revealed that there were different types of regulatory element for the Hina gene in wild and landrace/cultivated barleys. There were six consistent cis-acting binding sites in wild and landrace/cultivated barleys, whereas 8 to 16 inconsistent TATA-boxes were observed. In addition, three special elements (E2Fb, Sp1, and boxS) were only observed in wild barley, while one (AT1-motif) was only found in landrace/cultivated barley. Forty-four deduced amino acid sequences of HINA from wild and landrace/cultivated barleys were obtained by deleting repetitive amino acid sequences, and they were clustered into two groups on the basis of Neighbor-Joining analysis. However, there was no obvious difference in the amino acid sequences of HINA between wild and landrace/cultivated barleys. Comparing to protein secondary structure of wheat PINA, it was indicated that HINA also existed a signal peptide. In addition, HINA was a hydrophilic protein on the basis of the protein properties and composition.

There is a pressing need to identify therapeutic targets in tumors with low mutation rates such as the malignant pediatric brain tumor medulloblastoma. To address this challenge, we quantitatively profiled global proteomes and phospho-proteomes of 45 medulloblastoma samples. Integrated analyses revealed that tumors with similar RNA expression vary extensively at the post-transcriptional and post-translational levels. We identified distinct pathways associated with two subsets of SHH tumors, and found post-translational modifications of MYC that are associated with poor outcomes in group 3 tumors. We found kinases associated with subtypes and showed that inhibiting PRKDC sensitizes MYC-driven cells to radiation. Our study shows that proteomics enables a more comprehensive, functional readout, providing a foundation for future therapeutic strategies.

Primates have long been a favorite subject of evolutionary biologists, and in recent decades, have come to play an increasingly important role in biomedical research, including comparative genetics and phylogenetics. The growing list of annotated genome databases from nonhuman primate species are expected to aid in these endeavors, allowing many analyses to be performed partially or even entirely in silico. However, whole genome sequence data are typically derived from only one, or at best a few, individuals. As a consequence, information in the databases does not capture variation within species or populations, nor can the sequence of one individual be taken as representative across all loci. Furthermore, the vast majority of primate species have not been sequenced, and only a small percentage of species are currently slated for whole-genome sequencing efforts. Finally, for many species data on patterns and levels of RNA expression will be lacking. Thus, there will continue to be a demand for samples from nonhuman primates as raw material for genetic and phylogenetic analyses. Gathering such samples can be complicated, with many legal and practical barriers to obtaining samples in the field or transporting samples between research centers and across borders. Here, we provide basic but critical advice for those initiating studies requiring genetic material from nonhuman primates, including some guidance on how to locate and obtain samples, brief overviews of common protocols for handling and processing samples, and a table of useful links for locating resources related to the acquisition of samples. We also advocate for the creation of curated banks of nonhuman primate samples, particularly renewable sources of genetic material such as immortalized cell lines or fibroblasts, to reduce the need for repeated or redundant sampling from living animals.

Currently, comprehensive toxicological data are available only for a small percentage of the 30,000 substances produced in volumes of 1-100 tons per year in the EU. Substances with inadequate safety data sets may pose a risk to employees, consumers and the environment. To improve this unsatisfactory situation the European Commission put forward a draft concept that will probably become law in 2006. The acronym of this concept is REACH standing for Registration, Evaluation and Authorization of Chemicals. The aim of REACH is to systematically evaluate the risk of approximately 30,000 chemical substances produced, used or imported in quantities of 1-100 tons per year. From a practical point of view the testing requirements for these chemicals are one of the most important parts of the REACH proposal. The latter progressively increase with the volume of chemical substances, including, e.g. acute, subchronic and chronic toxicity tests. Without doubt REACH will provide an important contribution to health protection for workers and consumers. But perhaps even more importantly, REACH offers an opportunity to optimize and innovate testing strategies for chemicals. Such novel techniques are in particular RNA expression profiling, proteome analysis and metabonomics to describe alterations in gene or protein expressions patterns or in metabolite concentrations in response to toxic stimuli. Promising data have been published indicating that these techniques might identify hepato- or nephrotoxic compounds or even carcinogens differentiating between genotoxic and non-genotoxic substances. However, so far only a relatively small number of selected typical substances with well known toxic mechanisms has been tested. Therefore, the most promising innovative techniques should be optimized and validated by investigating a series of other typical but also untypical substances. In a further step a supplementary research program to REACH should be launched including promising innovative techniques (e.g. genomics, proteomics, metabonomics) but also other alternative methods (e.g. in vitro or QSAR), concentrating on the same substances that have to be tested by conventional animal studies in the mandatory part of REACH. In the present review we summarize key features of REACH, and discuss possibilities for the development of improved techniques and integrated strategies for toxicity testing.

Breast cancer is a major health problem in developed countries. Pathological and clinical heterogeneity, partly responsible of therapeutic failures, reflects its poorly documented complex and combinatory molecular basis. Thorough molecular typing could allow not only better tackling that diversity and improving the current prognostic classifications, but also could help in the identification of new molecular therapeutic targets. The recently developed DNA microarray technology allows the analysis of the RNA expression of several thousands of genes simultaneously in a sample. Recent studies have shown the promising prognostic impact of gene expression profiling in breast cancer by identifying new prognostic subclasses unidentifiable by conventional parameters.

Comparative gene-expression profiling is an important tool in understanding molecular signatures of complex diseases as well as the responses of cells and tissues to external factors. With increasing microarray data, disease-specific molecular patterns are emerging but the acquisition of these data is expensive, difficult to customize and not well standardized. Once genome-wide scans identify differentially expressed genes in a given disease, cheaper, more easily customized methods will be needed for evaluating the expression of these genes in large population samples. Here we describe a novel multiplex microsphere bead assay (MBA) to compare gene expression levels. To test this assay we evaluated the expression levels of four transcripts (BRCA1, MGB1, DLG1 and ACT1) in normal and cancerous mammary tissue. The results were consistent with those generated by quantitative real-time PCR.

Single cell analysis by flow cytometry is a powerful modality for analyzing the expression of a set of molecules. Nevertheless, the most significant deficiency of this technology has been the poor sensitivity of flow cytometry compared to other techniques such as immunoblotting. In order to address this deficiency, we have developed an enzymatic amplification system for flow cytometry that enhances the specific signal by 10-100-fold. Using enzymatic amplification staining (EAS), we and others have been able to assess the expression of molecules that could not be detected otherwise. In this review, we show the capability of this new technology to detect specific RNA expression.

High-throughput mRNA expression profiling is a widely used first step in the identification of new targets. Proper consideration of this step is essential before initiating the time-consuming and more elaborate biological validation of a target. This review discusses the strengths and limitations of this approach, with a special emphasis on studies in cardiac tissue. Specifically, it focuses on the basis of any such study, the tissue samples and the implications for statistical analysis of data retrieved from heterogeneous biological material. It also discusses studies performed in cardiac tissue and considers strategies to define target candidates based on expression profiles.

Background Molecular profiling assays have emerged as a promising tool in tumor diagnosis. Recent advances have allowed the use of formalin-fixed, paraffin-embedded (FFPE) tissues in such assays involving the use of microarrays. The Pathwork Tissue of Origin (TOO) Test was developed for use with FFPE tissue to aid tumor diagnosis. We sought to determine the performance of the TOO test on routine specimens from an independent laboratory. Methods Forty-five blinded, archived clinical specimens from the UCSF Department of Pathology were tested. Total RNA was processed to prepare labeled cDNA for hybridization to Pathchip microarrays. Hybridization data was analyzed with a 2000-gene classification model to quantify similarity between RNA expression of the study specimens and the 15 tissues on the test panel. Results 44/45 (98%) specimens were successfully processed. 37 cases met study inclusion criteria. Of these, 35 (95%) gave results which were in agreement with the reference diagnosis. In no case was the reference diagnosis ruled out. Conclusions The Tissue of Origin Test gave a high agreement with the reference diagnosis when archived clinical specimens from UCSF were assessed. Molecular profiling assays are highly accurate, and can be a useful tool in cancer diagnosis.

Expression of negative-strand murine coronavirus mouse hepatitis virus (MHV) defective interfering (DI) RNA transcripts in MHV-infected cells results in the accumulation of positive-strand DI RNAs (M. Joo et al., 1996, J. Virol. 70, 5769–5776). However, the expressed negative-strand DI RNA transcripts are poor templates for positive-strand DI RNA synthesis. The present study demonstrated that DI RNA accumulation from the expressed negative-strand DI RNA transcripts in MHV-infected cells was enhanced by the coexpression of complementary RNA transcripts that correspond to the 5′ region of positive-strand DI RNA. The positive-strand RNA transcripts corresponding to the 5′ end-most 0.7–2.0 kb DI RNA had a similar enhancement effect. The coexpressed positive-strand RNA transcripts lacking the leader sequence or those containing only the leader sequence failed to demonstrate this enhancement effect, demonstrating that the presence of the leader sequence in the coexpressed positive-strand RNA transcripts was necessary, but not sufficient, for the enhancement of DI RNA accumulation from the coexpressed negative-strand DI RNA transcripts. Negative-strand DI RNA transcripts that were coexpressed with the partial-length positive-strand RNA transcripts were no more stable than those expressed alone, suggesting that a higher stability of the expressed negative-strand RNA transcripts was an unlikely reason for the higher DI RNA accumulation in cells coexpressing two complementary DI RNA transcripts. Sequence analyses unexpectedly demonstrated that the leader sequence of the majority of accumulated DI RNAs switched to helper virus derived leader sequence, suggesting that enhancement of DI RNA accumulation was mediated by the efficient utilization of helper virus derived leader sequence for DI RNA synthesis. Furthermore, our data suggested that this leader switching, a type of homologous RNA–RNA recombination, occurred during positive-strand DI RNA synthesis and that MHV positive-strand RNA synthesis mechanism may have a preference toward recognizing double-stranded RNA structures over single-stranded negative-strand RNA to produce positive-strand DI RNAs.

The possibility that experimental data from diverse cell biology experiments might shed light on other experiments has been generally outside the realm of cancer biologists. Recent experiments suggest that core RNA expression profiles distilled from experiments using a set of known members with related attributes may be used as query tools to probe expression profiles from other unrelated experiments. The potential benefit arises from the possibility to share findings without fully reconstructing the exact initial conditions. The limitations will be framed by the robustness of the hypotheses so generated.

The Drosophila melanogaster trithorax gene encodes several large RNAs which are expressed in complex patterns in the embryo. The D. virilis trithorax gene was isolated and sequenced. It produces a similar to D. melanogaster set of transcripts, and it encodes a protein that shows sequence similarity in several domains which are also conserved in the human homologue, ALL-1 HRX . Previous experiments have suggested that a distinct expression domain of trithorax in the posterior region of the embryo is required to maintain expression of the BX-C genes (Sedkov et al., 1994, Development 120, 1907–1917). At cellular blastoderm, trithorax RNA expression in D. virilis embryos is also confined to the posterior portion of the presumptive mesoderm. This finding supports the idea that the specific BX-C-related expression domain is an essential feature of the trithorax gene.