A dual-reference study design for understanding and improving AAV genome size analysis.

Recombinant adeno-associated virus (rAAV) is the leading platform of gene delivery for its long-lasting gene transformation and low immunogenicity. Characterization of the integrity and purity of the rAAV genome is critical to ensure clinical potency and safety. However, current rAAV genome characterization methods that can provide size assessment are either time-consuming or not easily accessible to general labs. Additionally, there is a lack of right reference standard for analyzing long single-stranded DNA (ssDNA) fragments. Here, we have developed an ssDNA assay on a microfluidic capillary electrophoresis platform using ssDNA reference standard. This assay provides size calling for ssDNA fragment, a detection sensitivity at ∼89 pg/µL (3 × 1010  GC/mL AAV) for 5.1 kb ssDNA fragment, and a turnaround time at ∼100 s per sample with a high throughput sample analyzing capability. Moreover, we have observed that the annealing of AAV ssDNA subsequent to its release from the capsid might introduce an additional double-stranded DNA (dsDNA) peak. This phenomenon is dependent on the sample processing workflow. To avoid the risk of mischaracterization, we recommend the use of dual-reference standards in combination with other orthogonal methods to have a comprehensive understanding of the rAAV genome size and integrity.

Bioinspired Tumor Calcification-Guided Early Diagnosis and Eradication of Hepatocellular Carcinoma.

Tumor calcification is found to be associated with the benign prognostic, and which shows considerable promise as a somewhat predictive index of the tumor response clinically. However, calcification is still a missing area in clinical cancer treatment. A specific strategy is proposed for inducing tumor calcification through the synergy of calcium peroxide (CaO2)-based microspheres and transcatheter arterial embolization for the treatment of hepatocellular carcinoma (HCC). The persistent calcium stress in situ specifically leads to powerful tumor calcioptosis, resulting in diffuse calcification and a high-density shadow on computed tomography that enables clear localization of the in vivo tumor site and partial delineation of tumor margins in an orthotopic HCC rabbit model. This osmotic calcification can facilitate tumor clinical diagnosis, which is of great significance in differentiating tumor response during early follow-up periods. Proteome and phosphoproteome analysis identify that calreticulin (CALR) is a crucial target protein involved in tumor calcioptosis. Further fluorescence molecular imaging analysis also indicates that CALR can be used as a prodromal marker of calcification to predict tumor response at an earlier stage in different preclinical rodent models. These findings suggest that upregulated CALR in association with tumor calcification, which may be broadly useful for quick visualization of tumor response.

Internal Light Sources-Mediated Photodynamic Therapy Nanoplatforms: Hope for the Resolution of the Traditional Penetration Problem.

Photodynamic therapy (PDT) is an alternative cancer treatment technique with a noninvasive nature, high selectivity, and minimal adverse effects. The indispensable light source used in PDT is a critical factor in determining the energy conversion of photosensitizers (PSs). Traditional light sources are primarily concentrated in the visible light region, severely limiting their penetration depth and making them prone to scattering and absorption when applied to biological tissues. For that reason, its efficacy in treating deep-seated lesions is often inadequate. Self-exciting PDT, also known as auto-PDT (APDT), is an attractive option for circumventing the limited penetration depth of traditional PDT and has acquired significant attention. APDT employs depth-independent internal light sources to excite PSs through resonance or radiative energy transfer. APDT has considerable potential for treating deep-tissue malignancies. To facilitate many researchers' comprehension of the latest research progress in this field and inspire the emergence of more novel research results. This review introduces internal light generation mechanisms and characteristics and provides an overview of current research progress based on the recently reported APDT nanoplatforms. The current challenges and possible solutions of APDT nanoplatforms are also presented and provide insights for future research in the final section of this article.

Molecular Engineering of NIR-II/IIb Emitting AIEgen for Multimodal Imaging-Guided Photo-Immunotherapy.

In view of the great challenges related to the complexity and heterogeneity of tumors, efficient combination therapy is an ideal strategy for eliminating primary tumors and inhibiting distant tumors. A novel aggregation-induced emission (AIE) phototherapeutic agent called T-TBBTD is developed, which features a donor-acceptor-donor (D-A-D) structure, enhanced twisted molecule conformation, and prolonged second near-infrared window (NIR-II) emission. The multimodal imaging function of the molecule has significance for its treatment time window and excellent photothermal/photodynamic performance for multimode therapy. The precise molecular structure and versatility provide prospects for molecular therapy for anti-tumor applications. Fluorescence imaging in the NIR-II window offers advantages with enhanced spatial resolution, temporal resolution, and penetration depth. The prepared AIE@R837 NPs also have controllable performance for antitumor photo-immunotherapy. Following local photo-irradiation, AIE@R837 NPs generate abundant heat, and 1 O2 directly kills tumor cells, induces immunogenic cell death (ICD) as a photo-therapeutic effect, and releases R837, which enhances the synergistic effect of antigen presentation and contributes to the long-lasting protective antitumor immunity. A bilateral 4T1 tumor model revealed that this photo-immunotherapy can eliminate primary tumors. More importantly, it has a significant inhibitory effect on distant tumor growth. Therefore, this method can provide a new strategy for tumor therapy.

Integrated Nanorod-Mediated PD-L1 Downregulation in Combination with Oxidative-Stress Immunogene Therapy against Cancer.

It is an engaging program for tumor treatment that rationalizes the specific microenvironments, activation of suppressed immune system (immune resistance/escape reversion), and synergistic target therapy. Herein, a biomimetic nanoplatform that combines oxidative stress with genetic immunotherapy to strengthen the therapeutic efficacy is developed. Ru-TePt nanorods, small interfering RNA (PD-L1 siRNA), and biomimetic cellular membrane vesicles with the targeting ability to design a multifunctional Ru-TePt@siRNA-MVs system are rationally integrated. Notably, the Fenton-like activity significantly enhances Ru-TePt nanorods sonosensitization, thus provoking stronger oxidative stress to kill cells directly. Meanwhile, immunogenic cell death is triggered to secrete numerous cytokines and activate T cells. The effective catalase characteristics of Ru-TePt enable the in situ oxygen-producing pump to improve tumor oxygen level and coordinately strengthen the therapeutic effect of SDT followed. More importantly, anti-PD-L1-siRNA mediated immune checkpoint silence of the PD-L1 gene creates an environment conducive to activating cytotoxic T lymphocytes, synergistic with boosted reactive oxygen species-triggered antitumor immune response. The experimental results in vitro and in vivo reveal that the Ru-TePt@siRNA-MVs nanosystems can effectively activate the oxidative stress-triggered immune response and inhibit PD-1/PD-L1 axis-mediated immune resistance. Consequently, this orchestrated treatment paradigm provides valuable insights for developing potential oxidative stress and genetic immunotherapy.

Broadening the Horizons of RNA Delivery Strategies in Cancer Therapy.

RNA-based therapy is a promising and innovative strategy for cancer treatment. However, poor stability, immunogenicity, low cellular uptake rate, and difficulty in endosomal escape are considered the major obstacles in the cancer therapy process, severely limiting the development of clinical translation and application. For efficient and safe transport of RNA into cancer cells, it usually needs to be packaged in appropriate carriers so that it can be taken up by the target cells and then be released to the specific location to perform its function. In this review, we will focus on up-to-date insights of the RNA-based delivery carrier and comprehensively describe its application in cancer therapy. We briefly discuss delivery obstacles in RNA-mediated cancer therapy and summarize the advantages and disadvantages of different carriers (cationic polymers, inorganic nanoparticles, lipids, etc.). In addition, we further summarize and discuss the current RNA therapeutic strategies approved for clinical use. A comprehensive overview of various carriers and emerging delivery strategies for RNA delivery, as well as the current status of clinical applications and practice of RNA medicines are classified and integrated to inspire fresh ideas and breakthroughs.

Amplifying Dendritic Cell Activation by Bioinspired Nanometal Organic Frameworks for Synergistic Sonoimmunotherapy.

Despite recent advancements of sonodynamic therapy (SDT) in cancer immunotherapy, challenges have yet to be surmounted to further boost its immunotherapeutic efficacy due to the low-level tumor antigens presentation of dendritic cells (DCs). Cell membrane camouflaged-nanoparticles can integrate the neoantigens of the cancer cell membrane with the multifunctionalities of synthetic nanocores. Herein, sono-responsive nanoparticles coated with DC-targeted antibody chimeric cancer cell membrane are investigated for multimodal therapy. The nanometal organic frameworks (MOFs) that respond to ultrasound are loaded successfully inside the vesicles displaying an anti-DEC205 antibody. The anti-DEC205 chimeric vesicles can directly target and activate DCs, promote tumor antigens cross-presentation, and then produce a cascade amplified T-cell immune response. Upon deep tissue-penetrating sonication, AMR-MOF@AuPt generates large amounts of reactive oxygen species that directly kill cancer cells, further initiating an anti-cancer T cell immune response. Such synergistic sono-immunotherapies effectually inhibit tumor growth and induce strong systemic and long-term immune memory against cancer recurrence and distant metastasis. The authors findings provide DCs and tumor cells of a dual active-targeting cell membrane-coated sono-immunotherapeutic nanoplatform for cancer therapy.

Connecting Calcium-Based Nanomaterials and Cancer: From Diagnosis to Therapy.

As the indispensable second cellular messenger, calcium signaling is involved in the regulation of almost all physiological processes by activating specific target proteins. The importance of calcium ions (Ca2+) makes its "Janus nature" strictly regulated by its concentration. Abnormal regulation of calcium signals may cause some diseases; however, artificial regulation of calcium homeostasis in local lesions may also play a therapeutic role. "Calcium overload," for example, is characterized by excessive enrichment of intracellular Ca2+, which irreversibly switches calcium signaling from "positive regulation" to "reverse destruction," leading to cell death. However, this undesirable death could be defined as "calcicoptosis" to offer a novel approach for cancer treatment. Indeed, Ca2+ is involved in various cancer diagnostic and therapeutic events, including calcium overload-induced calcium homeostasis disorder, calcium channels dysregulation, mitochondrial dysfunction, calcium-associated immunoregulation, cell/vascular/tumor calcification, and calcification-mediated CT imaging. In parallel, the development of multifunctional calcium-based nanomaterials (e.g., calcium phosphate, calcium carbonate, calcium peroxide, and hydroxyapatite) is becoming abundantly available. This review will highlight the latest insights of the calcium-based nanomaterials, explain their application, and provide novel perspective. Identifying and characterizing new patterns of calcium-dependent signaling and exploiting the disease element linkage offer additional translational opportunities for cancer theranostics.

Recent Advancements in Poor Graft Function Following Hematopoietic Stem Cell Transplantation.

Poor graft function (PGF) is a life-threatening complication that occurs after transplantation and has a poor prognosis. With the rapid development of haploidentical hematopoietic stem cell transplantation, the pathogenesis of PGF has become an important issue. Studies of the pathogenesis of PGF have resulted in some success in CD34+-selected stem cell boosting. Mesenchymal stem cells, N-acetyl-l-cysteine, and eltrombopag have also been investigated as therapeutic strategies for PGF. However, predicting and preventing PGF remains challenging. Here, we propose that the seed, soil, and insect theories of aplastic anemia also apply to PGF; CD34+ cells are compared to seeds; the bone marrow microenvironment to soil; and virus infection, iron overload, and donor-specific anti-human leukocyte antigen antibodies to insects. From this perspective, we summarize the available information on the common risk factors of PGF, focusing on its potential mechanism. In addition, the safety and efficacy of new strategies for treating PGF are discussed to provide a foundation for preventing and treating this complex clinical problem.

Endoscopic modified total laminoplasty for symptomatic lumbar spinal stenosis.

Context/objective: At present, there is no consensus on the most effective surgical method for treating symptomatic lumbar spinal stenosis (LSS). Total laminectomy, which is frequently used at this time, destroys the posterior midline structure, causing many postoperative complications. We have designed a new surgical approach instead of total laminectomy. In this paper, we aimed to describe the surgical method of endoscopic modified total laminectomy for lumbar spinal stenosis as well as to explore its early efficacy.Participants: Patients with symptomatic LSS who underwent endoscopic modified total laminoplasty between August 2016 and August 2017 were eligible for our study.Outcome measures: Before surgery and one year after surgery, we measured lower limb pain and back pain by visual analog scale (VAS), disability via Oswestry Disability Index (ODI), and severity of back pain according to the Japanese Orthopedic Association Score for Back Pain (JOA), while any complications were also assessed.Results: Endoscopic modified total laminoplasty was performed on 22 LSS patients, including eight males and 14 females(mean age = 59.3 ± 9.6 years). We found statistically significant differences before and one year after surgery for VAS lower limb pain and back pain, ODI and JOA scores(P < 0.001). Complications included intraoperative dural tears(n = 1),and weak fusion between the lamina and the vertebral body (n = 1).Conclusion: Endoscopic modified total laminectomy is a promising surgical approach which reduces patient suffering and improves patient quality of life.

Nanotransferrin-Based Programmable Catalysis Mediates Three-Pronged Induction of Oxidative Stress to Enhance Cancer Immunotherapy.

Current oxidative stress amplifying strategies for immunogenic cell death (ICD) promotion are mainly restricted to immune tolerance induced by adaptive cellular antioxidation, limited tumor-selectivity, and tumoral immunosuppression. Herein, a facile and efficient scenario of genetically engineering transferrin-expressing cell membrane nanovesicle encapsulated IR820-dihydroartemisinin nanomedicine (Tf@IR820-DHA) was developed to boost a-PD-L1-mediated immune checkpoint blocking (ICB) via synergetic triple stimuli-activated oxidative stress-associated ICD. We demonstrate that the engineered transferrin of Tf@IR820-DHA has excellent tumor targeting and Fe(III)-loading properties and thus delivered Fe(III) and IR820-DHA nanoparticles (NPs) to the lesion location effectively. We found that the self-carrying Fe(III)-mediated programmable catalysis of DHA and glutathione (GSH) depletion generated plenty of reactive oxygen species (ROS). Moreover, DHA also acted as an immunomodulator to decrease the number of T regulatory cells, thereby remodeling the tumor immune microenvironment and achieving double T cell activation. Furthermore, the IR820 molecule served as a competent sonosensitizer to produce ROS under ultrasound activation and guide precise immunotherapy via fluorescent/photoacoustic (FL/PA) imaging. Through its three-pronged delivery of stimuli-activated oxidative stress (DHA-induced chemodynamic therapy, catalysis-conferred GSH depletion, and IR820-mediated sonodynamic therapy), Tf@IR820-DHA caused high levels of targeted ICD. This significantly increased the proportions of IFN-γ-secreting T cells (CD4+ T and CD8+ T) and enhanced a-PD-L1-mediated ICB against primary and distant tumors, which represents a promising approach for cancer nanoimmunotherapy.

Biomimetic nanoparticles blocking autophagy for enhanced chemotherapy and metastasis inhibition via reversing focal adhesion disassembly.

BACKGROUND: Autophagy is a conserved catabolic process, which plays an important role in regulating tumor cell motility and degrading protein aggregates. Chemotherapy-induced autophagy may lead to tumor distant metastasis and even chemo-insensitivity in the therapy of hepatocellular carcinoma (HCC). Therefore, a vast majority of HCC cases do not produce a significant response to monotherapy with autophagy inhibitors. RESULTS: In this work, we developed a biomimetic nanoformulation (TH-NP) co-encapsulating Oxaliplatin (OXA)/hydroxychloroquine (HCQ, an autophagy inhibitor) to execute targeted autophagy inhibition, reduce tumor cell migration and invasion in vitro and attenuate metastasis in vivo. The tumor cell-specific ligand TRAIL was bioengineered to be stably expressed on HUVECs and the resultant membrane vesicles were wrapped on OXA/HCQ-loaded PLGA nanocores. Especially, TH-NPs could significantly improve OXA and HCQ effective concentration by approximately 21 and 13 times in tumor tissues compared to the free mixture of HCQ/OXA. Moreover, the tumor-targeting TH-NPs released HCQ alkalized the acidic lysosomes and inhibited the fusion of autophagosomes and lysosomes, leading to effective blockade of autophagic flux. In short, the system largely improved chemotherapeutic performance of OXA on subcutaneous and orthotopic HCC mice models. Importantly, TH-NPs also exhibited the most effective inhibition of tumor metastasis in orthotopic HCCLM3 models, and in the HepG2, Huh-7 or HCCLM3 metastatic mice models. Finally, we illustrated the enhanced metastasis inhibition was attributed to the blockade or reverse of the autophagy-mediated degradation of focal adhesions (FAs) including E-cadherin and paxillin. CONCLUSIONS: TH-NPs can perform an enhanced chemotherapy and antimetastatic effect, and may represent a promising strategy for HCC therapy in clinics.

Elicitor hydrophobin Hyd1 interacts with Ubiquilin1-like to induce maize systemic resistance.

Trichoderma harzianum is a plant-beneficial fungus that secretes small cysteine-rich proteins that induce plant defense responses; however, the molecular mechanism involved in this induction is largely unknown. Here, we report that the class II hydrophobin ThHyd1 acts as an elicitor of induced systemic resistance (ISR) in plants. Immunogold labeling and immunofluorescence revealed ThHyd1 localized on maize (Zea mays) root cell plasma membranes. To identify host plant protein interactors of Hyd1, we screened a maize B73 root cDNA library. ThHyd1 interacted directly with ubiquilin 1-like (UBL). Furthermore, the N-terminal fragment of UBL was primarily responsible for binding with Hyd1 and the eight-cysteine amino acid of Hyd1 participated in the protein-protein interactions. Hyd1 from T. harzianum (Thhyd1) and ubl from maize were co-expressed in Arabidopsis thaliana, they synergistically promoted plant resistance against Botrytis cinerea. RNA-sequencing analysis of global gene expression in maize leaves 24 h after spraying with Curvularia lunata spore suspension showed that Thhyd1-induced systemic resistance was primarily associated with brassinosteroid signaling, likely mediated through BAK1. Jasmonate/ethylene (JA/ET) signaling was also involved to some extent in this response. Our results suggest that the Hyd1-UBL axis might play a key role in inducing systemic resistance as a result of Trichoderma-plant interactions.

[Impact of JAK2V617F Mutation Burden on Clinial Presentation and Survival in ET Patients].

OBJECTIVE: To better define the effect of JAK2V617F mutant allele burden on clinical presentation of patients with essential thrombo cythamia (ET), especially thrombosis. METHODS: Two ml of heparin anti-coagulated bone marrow was collected from 229 ET cases, who were diagnosed and treated in the First People's Hospital of Yunnan Province during 2013.10 to 2016.12. and then the mononuclear cells were separated by Red Blood Cell Lysis Buffer, genomic DNA was extracted from mononuclear cells by using a commercial DNA isolation kit and amplified by allele specific polymerase chain reaction (PCR). According to the size of molecular weight, the amplified products were separated by electrophoresis on a 2% agarose gel to screen the JAK2V617F mutation, then the JAK2V617F mutation burden was detected by real-time polymerase chain reaction (RT-PCR) in 120 patients with JAK2V617F mutation. Meanwhile, these samples were sequenced in order to verify the accuracy of the PCR screewing. RESULTS: ET patients with thrombotic events had significantly higher JAK2V617F allele burden than those without thrombosis (23.2% vs 14.2%) ( P<0.05). Meanwhile, ET patients showed increased JAK2V617F allele burden in the group with higher leukocytosis (WBC > 10×109/L) (P<0.001) and hemoglobin (> 150 g/L) (P<0.05). JAK2V617F mutation burden in 17 patients with splenomegaly was higher than that in 45 patients without splenomegaly (28.1% vs 11.8%) (P<0.05). but the JAK2V617F mutation burden was regatively correlated with platelet count (P<0.05). On the other hand, no correlation was found between JAK2V617F mutation burden and sex (P > 0.05). Univariate analysis showed that the JAK2V617F allele burden did not affect survival. Multivariable analysis showed that prognostic variable including WBC counts, hemoglobin level, age, sex, and splenomegaly not affected survival, (P > 0.05). CONCLUSION: The clinical presentations of ET patients, such as WBC counts, hemoglobin level and splenomegaly, are influenced by the JAK2V617F mutation burden. ET patients with thrombotic events has significantly higher JAK2V617F allele burden than those in ET palients without thrombosis.JAK2V617F mutation burden has no relations with sex and age..

Optimal donor for severe aplastic anemia patient requiring allogeneic hematopoietic stem cell transplantation: A large-sample study from China.

HLA-haploidentical hematopoietic stem cell transplantation (HSCT) may be an option for severe aplastic anemia (SAA) patients. However, to date, no large-sample studies have been performed to determine which types of SAA patients are suitable for HLA-haploidentical HSCT. We retrospectively studied 189 consecutive patients with SAA who underwent HLA-identical or HLA-haploidentical HSCT at seven transplant centers in China. Propensity score matching (PSM) was applied in this study to reduce the influence of potential confounders. The 5-year overall survival (OS) rate was 72.0% in the HLA-haploidentical group and 76.5% in the HLA-identical group. The median time to achieve engraftment and the incidence of acute GVHD/chronic GVHD were not significantly different between the two groups. In the subgroup analysis, the outcome of patients older than 40 years in the HLA-haploidentical group was significantly poorer than that of patients younger than 40 years in the same group and that of patients older than 40 years in the HLA-identical group. Based on the above results, we suggest that HLA-haploidentical relative HSCT should be considered as a valid alternative option for patients younger than 40 years with SAA for whom no matched sibling donor is available.

Concerted effects of heterogeneous nuclear ribonucleoprotein C1/C2 to control vitamin D-directed gene transcription and RNA splicing in human bone cells.

Traditionally recognized as an RNA splicing regulator, heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC1/C2) can also bind to double-stranded DNA and function in trans as a vitamin D response element (VDRE)-binding protein. As such, hnRNPC1/C2 may couple transcription induced by the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D) with subsequent RNA splicing. In MG63 osteoblastic cells, increased expression of the 1,25(OH)2D target gene CYP24A1 involved immunoprecipitation of hnRNPC1/C2 with CYP24A1 chromatin and RNA. Knockdown of hnRNPC1/C2 suppressed expression of CYP24A1, but also increased expression of an exon 10-skipped CYP24A1 splice variant; in a minigene model the latter was attenuated by a functional VDRE in the CYP24A1 promoter. In genome-wide analyses, knockdown of hnRNPC1/C2 resulted in 3500 differentially expressed genes and 2232 differentially spliced genes, with significant commonality between groups. 1,25(OH)2D induced 324 differentially expressed genes, with 187 also observed following hnRNPC1/C2 knockdown, and a further 168 unique to hnRNPC1/C2 knockdown. However, 1,25(OH)2D induced only 10 differentially spliced genes, with no overlap with differentially expressed genes. These data indicate that hnRNPC1/C2 binds to both DNA and RNA and influences both gene expression and RNA splicing, but these actions do not appear to be linked through 1,25(OH)2D-mediated induction of transcription.

m(6)A-LAIC-seq reveals the census and complexity of the m(6)A epitranscriptome.

N(6)-Methyladenosine (m(6)A) is a widespread, reversible chemical modification of RNA molecules, implicated in many aspects of RNA metabolism. Little quantitative information exists as to either how many transcript copies of particular genes are m(6)A modified ('m(6)A levels') or the relationship of m(6)A modification(s) to alternative RNA isoforms. To deconvolute the m(6)A epitranscriptome, we developed m(6)A-level and isoform-characterization sequencing (m(6)A-LAIC-seq). We found that cells exhibit a broad range of nonstoichiometric m(6)A levels with cell-type specificity. At the level of isoform characterization, we discovered widespread differences in the use of tandem alternative polyadenylation (APA) sites by methylated and nonmethylated transcript isoforms of individual genes. Strikingly, there is a strong bias for methylated transcripts to be coupled with proximal APA sites, resulting in shortened 3' untranslated regions, while nonmethylated transcript isoforms tend to use distal APA sites. m(6)A-LAIC-seq yields a new perspective on transcriptome complexity and links APA usage to m(6)A modifications.

The contribution of Alu exons to the human proteome.

BACKGROUND: Alu elements are major contributors to lineage-specific new exons in primate and human genomes. Recent studies indicate that some Alu exons have high transcript inclusion levels or tissue-specific splicing profiles, and may play important regulatory roles in modulating mRNA degradation or translational efficiency. However, the contribution of Alu exons to the human proteome remains unclear and controversial. The prevailing view is that exons derived from young repetitive elements, such as Alu elements, are restricted to regulatory functions and have not had adequate evolutionary time to be incorporated into stable, functional proteins. RESULTS: We adopt a proteotranscriptomics approach to systematically assess the contribution of Alu exons to the human proteome. Using RNA sequencing, ribosome profiling, and proteomics data from human tissues and cell lines, we provide evidence for the translational activities of Alu exons and the presence of Alu exon derived peptides in human proteins. These Alu exon peptides represent species-specific protein differences between primates and other mammals, and in certain instances between humans and closely related primates. In the case of the RNA editing enzyme ADARB1, which contains an Alu exon peptide in its catalytic domain, RNA sequencing analyses of A-to-I editing demonstrate that both the Alu exon skipping and inclusion isoforms encode active enzymes. The Alu exon derived peptide may fine tune the overall editing activity and, in limited cases, the site selectivity of ADARB1 protein products. CONCLUSIONS: Our data indicate that Alu elements have contributed to the acquisition of novel protein sequences during primate and human evolution.

Transcriptome-wide landscape of pre-mRNA alternative splicing associated with metastatic colonization.

UNLABELLED: Metastatic colonization is an ominous feature of cancer progression. Recent studies have established the importance of pre-mRNA alternative splicing (AS) in cancer biology. However, little is known about the transcriptome-wide landscape of AS associated with metastatic colonization. Both in vitro and in vivo models of metastatic colonization were utilized to study AS regulation associated with cancer metastasis. Transcriptome profiling of prostate cancer cells and derivatives crossing in vitro or in vivo barriers of metastasis revealed splicing factors with significant gene expression changes associated with metastatic colonization. These include splicing factors known to be differentially regulated in epithelial-mesenchymal transition (ESRP1, ESRP2, and RBFOX2), a cellular process critical for cancer metastasis, as well as novel findings (NOVA1 and MBNL3). Finally, RNA-seq indicated a large network of AS events regulated by multiple splicing factors with altered gene expression or protein activity. These AS events are enriched for pathways important for cell motility and signaling, and affect key regulators of the invasive phenotype such as CD44 and GRHL1. IMPLICATIONS: Transcriptome-wide remodeling of AS is an integral regulatory process underlying metastatic colonization, and AS events affect the metastatic behavior of cancer cells.

rMATS: robust and flexible detection of differential alternative splicing from replicate RNA-Seq data.

Ultra-deep RNA sequencing (RNA-Seq) has become a powerful approach for genome-wide analysis of pre-mRNA alternative splicing. We previously developed multivariate analysis of transcript splicing (MATS), a statistical method for detecting differential alternative splicing between two RNA-Seq samples. Here we describe a new statistical model and computer program, replicate MATS (rMATS), designed for detection of differential alternative splicing from replicate RNA-Seq data. rMATS uses a hierarchical model to simultaneously account for sampling uncertainty in individual replicates and variability among replicates. In addition to the analysis of unpaired replicates, rMATS also includes a model specifically designed for paired replicates between sample groups. The hypothesis-testing framework of rMATS is flexible and can assess the statistical significance over any user-defined magnitude of splicing change. The performance of rMATS is evaluated by the analysis of simulated and real RNA-Seq data. rMATS outperformed two existing methods for replicate RNA-Seq data in all simulation settings, and RT-PCR yielded a high validation rate (94%) in an RNA-Seq dataset of prostate cancer cell lines. Our data also provide guiding principles for designing RNA-Seq studies of alternative splicing. We demonstrate that it is essential to incorporate biological replicates in the study design. Of note, pooling RNAs or merging RNA-Seq data from multiple replicates is not an effective approach to account for variability, and the result is particularly sensitive to outliers. The rMATS source code is freely available at rnaseq-mats.sourceforge.net/. As the popularity of RNA-Seq continues to grow, we expect rMATS will be useful for studies of alternative splicing in diverse RNA-Seq projects.