R-Loops in Genome Instability and Cancer.

R-loops are unique, three-stranded nucleic acid structures that primarily form when an RNA molecule displaces one DNA strand and anneals to the complementary DNA strand in a double-stranded DNA molecule. R-loop formation can occur during natural processes, such as transcription, in which the nascent RNA molecule remains hybridized with the template DNA strand, while the non-template DNA strand is displaced. However, R-loops can also arise due to many non-natural processes, including DNA damage, dysregulation of RNA degradation pathways, and defects in RNA processing. Despite their prevalence throughout the whole genome, R-loops are predominantly found in actively transcribed gene regions, enabling R-loops to serve seemingly controversial roles. On one hand, the pathological accumulation of R-loops contributes to genome instability, a hallmark of cancer development that plays a role in tumorigenesis, cancer progression, and therapeutic resistance. On the other hand, R-loops play critical roles in regulating essential processes, such as gene expression, chromatin organization, class-switch recombination, mitochondrial DNA replication, and DNA repair. In this review, we summarize discoveries related to the formation, suppression, and removal of R-loops and their influence on genome instability, DNA repair, and oncogenic events. We have also discussed therapeutical opportunities by targeting pathological R-loops.

Fanconi anemia associated protein 20 (FAAP20) plays an essential role in homology-directed repair of DNA double-strand breaks.

FAAP20 is a Fanconi anemia (FA) protein that associates with the FA core complex to promote FANCD2/FANCI monoubiquitination and activate the damage response to interstrand crosslink damage. Here, we report that FAAP20 has a marked role in homologous recombination at a DNA double-strand break not associated with an ICL and separable from its binding partner FANCA. While FAAP20's role in homologous recombination is not dependent on FANCA, we found that FAAP20 stimulates FANCA's biochemical activity in vitro and participates in the single-strand annealing pathway of double-strand break repair in a FANCA-dependent manner. This indicates that FAAP20 has roles in several homology-directed repair pathways. Like other homology-directed repair factors, FAAP20 loss causes a reduction in nuclear RAD51 Irradiation-induced foci; and sensitizes cancer cells to ionizing radiation and PARP inhibition. In summary, FAAP20 participates in DNA double strand break repair by supporting homologous recombination in a non-redundant manner to FANCA, and single-strand annealing repair via FANCA-mediated strand annealing activity.

Integrative network-based analysis on multiple Gene Expression Omnibus datasets identifies novel immune molecular markers implicated in non-alcoholic steatohepatitis.

Introduction: Non-alcoholic steatohepatitis (NASH), an advanced subtype of non-alcoholic fatty liver disease (NAFLD), has becoming the most important aetiology for end-stage liver disease, such as cirrhosis and hepatocellular carcinoma. This study were designed to explore novel genes associated with NASH. Methods: Here, five independent Gene Expression Omnibus (GEO) datasets were combined into a single cohort and analyzed using network biology approaches. Results: 11 modules identified by weighted gene co-expression network analysis (WGCNA) showed significant association with the status of NASH. Further characterization of four gene modules of interest demonstrated that molecular pathology of NASH involves the upregulation of hub genes related to immune response, cholesterol and lipid metabolic process, extracellular matrix organization, and the downregulation of hub genes related to cellular amino acid catabolic, respectively. After DEGs enrichment analysis and module preservation analysis, the Turquoise module associated with immune response displayed a remarkably correlation with NASH status. Hub genes with high degree of connectivity in the module, including CD53, LCP1, LAPTM5, NCKAP1L, C3AR1, PLEK, FCER1G, HLA-DRA and SRGN were further verified in clinical samples and mouse model of NASH. Moreover, single-cell RNA-seq analysis showed that those key genes were expressed by distinct immune cells such as microphages, natural killer, dendritic, T and B cells. Finally, the potential transcription factors of Turquoise module were characterized, including NFKB1, STAT3, RFX5, ILF3, ELF1, SPI1, ETS1 and CEBPA, the expression of which increased with NASH progression. Discussion: In conclusion, our integrative analysis will contribute to the understanding of NASH and may enable the development of potential biomarkers for NASH therapy.

A convenient polyculture system that controls a shrimp viral disease with a high transmission rate.

Developing ecological approaches for disease control is critical for future sustainable aquaculture development. White spot syndrome (WSS), caused by white spot syndrome virus (WSSV), is the most severe disease in cultured shrimp production. Culturing specific pathogen-free (SPF) broodstock is an effective and widely used strategy for controlling WSS. However, most small-scale farmers, who predominate shrimp aquaculture in developing countries, cannot cultivate SPF shrimp, as they do not have the required infrastructure and skills. Thus, these producers are more vulnerable to WSS outbreaks than industrial farms. Here we developed a shrimp polyculture system that prevents WSS outbreaks by introducing specific fish species. The system is easy to implement and requires no special biosecurity measures. The promotion of this system in China demonstrated that it allowed small-scale farmers to improve their livelihood through shrimp cultivation by controlling WSS outbreaks and increasing the production of ponds.

Sub-pixel position estimation algorithm based on Gaussian fitting and sampling theorem interpolation for wafer alignment.

Wafer alignment is the core technique of lithographic tools. Image-processing-based wafer alignment techniques are commonly used in lithographic tools. An alignment algorithm is used to analyze the alignment mark image for obtaining the mark position. The accuracy and speed of the alignment algorithm are very important for guaranteeing the overlay and throughput of lithographic tools. The most commonly used algorithm in image-processing-based alignment techniques is the self-correlation method. This method has a high accuracy, but the calculation is complex, and the calculation speed is slow. In this paper, we propose a sub-pixel position estimation algorithm based on Gaussian fitting and sampling theorem interpolation. The algorithm first reconstructs the alignment signal by sampling theorem interpolation and then obtains the sub-pixel position of the mark by Gaussian fitting. The accuracy and robustness of the algorithm are verified by testing the simulated marks and experimentally captured marks. The repeat accuracy can reach 1/100 pixels, which is in the same level with the self-correlation method. The calculation speed is highly improved compared with the self-correlation method, which needs only about 1/3 of even short calculation time.

The Polycomb protein RING1B enables estrogen-mediated gene expression by promoting enhancer-promoter interaction and R-loop formation.

Polycomb complexes have traditionally been prescribed roles as transcriptional repressors, though increasing evidence demonstrate they can also activate gene expression. However, the mechanisms underlying positive gene regulation mediated by Polycomb proteins are poorly understood. Here, we show that RING1B, a core component of Polycomb Repressive Complex 1, regulates enhancer-promoter interaction of the bona fide estrogen-activated GREB1 gene. Systematic characterization of RNA:DNA hybrid formation (R-loops), nascent transcription and RNA Pol II activity upon estrogen administration revealed a key role of RING1B in gene activation by regulating R-loop formation and RNA Pol II elongation. We also found that the estrogen receptor alpha (ERα) and RNA are both necessary for full RING1B recruitment to estrogen-activated genes. Notably, RING1B recruitment was mostly unaffected upon RNA Pol II depletion. Our findings delineate the functional interplay between RING1B, RNA and ERα to safeguard chromatin architecture perturbations required for estrogen-mediated gene regulation and highlight the crosstalk between steroid hormones and Polycomb proteins to regulate oncogenic programs.

Measurement algorithm for wafer alignment based on principal component analysis.

We propose a novel measurement algorithm for wafer alignment technology based on principal component analysis (PCA) of a mark image. The waveform of the mark is extracted from the enlarged mark image, which is collected by CCD. The position of the mark center on the CCD can be calculated based on the extracted waveform. By applying PCA to the mark image, the first principal component containing position information of the mark can be obtained. Therefore PCA can be used to extract the waveform from the mark image. Compared with the typical waveform extraction method (the summed projection (SP) method), the proposed PCA method can use the position information contained in the mark image more effectively. Through simulation and experiment, it is proved that the proposed PCA method can improve the contrast of the normalized waveform, and then improve the alignment accuracy.

Fanconi anemia pathway as a prospective target for cancer intervention.

Fanconi anemia (FA) is a recessive genetic disorder caused by biallelic mutations in at least one of 22 FA genes. Beyond its pathological presentation of bone marrow failure and congenital abnormalities, FA is associated with chromosomal abnormality and genomic instability, and thus represents a genetic vulnerability for cancer predisposition. The cancer relevance of the FA pathway is further established with the pervasive occurrence of FA gene alterations in somatic cancers and observations of FA pathway activation-associated chemotherapy resistance. In this article we describe the role of the FA pathway in canonical interstrand crosslink (ICL) repair and possible contributions of FA gene alterations to cancer development. We also discuss the perspectives and potential of targeting the FA pathway for cancer intervention.

The Applications of Nanopore Sequencing Technology in Pathogenic Microorganism Detection.

Infectious diseases are major threats to human health and lead to a serious public health burden. The emergence of new pathogens and the mutation of known pathogens challenge our ability to diagnose and control infectious diseases. Nanopore sequencing technology exhibited versatile applications in pathogenic microorganism detection due to its flexible data throughput. This review article introduced the applications of nanopore sequencing in clinical microbiology and infectious diseases management, including the monitoring of emerging infectious diseases outbreak, identification of pathogen drug resistance, and disease-related microbial communities characterization.

microRNA-30a inhibits the liver cell proliferation and promotes cell apoptosis through the JAK/STAT signaling pathway by targeting SOCS-1 in rats with sepsis.

Sepsis is a systemic inflammatory response that may be induced by trauma, infection, surgery, and burns. With the aim of discovering novel treatment targets for sepsis, this current study was conducted to investigate the effect and potential mechanism by which microRNA-30a (miR-30a) controls sepsis-induced liver cell proliferation and apoptosis. Rat models of sepsis were established by applying the cecal ligation and puncture (CLP) method to simulate sepsis models. The binding site between miR-30a and suppressor of cytokine signaling protein 1 (SOCS-1) was determined by dual luciferase reporter gene assay. The gain-of-and-loss-of-function experiments were applied to analyze the effects of miR-30a and SOCS-1 on liver cell proliferation and apoptosis of the established sepsis rat models. The expression of miR-30a, SOCS-1, Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), Bcl-2 associated X protein (Bax), B cell lymphoma-2 (Bcl-2), toll-like receptor 4 (TLR4), and high-mobility group box 1 (HMGB1), and the extent of JAK2 and STAT3 phosphorylation were all determined. Sepsis led to an elevation of miR-30a and also a decline of SOCS-1 in the liver cells. SOCS-1 was negatively regulated by miR-30a. Upregulated miR-30a and downregulated SOCS-1 increased the expression of JAK2, STAT3, Bax, TLR4, and HMGB1 as well as the extent of JAK2 and STAT3 phosphorylation whereas impeding the expression of SOCS-1 and Bcl-2. More important, either miR-30a elevation or SOCS-1 silencing suppressed liver cell proliferation and also promoted apoptosis. On the contrary, the inhibition of miR-30a exhibited the opposite effects. Altogether, we come to the conclusion that miR-30a inhibited the liver cell proliferation and promoted cell apoptosis by targeting and negatively regulating SOCS-1 via the JAK/STAT signaling pathway in rats with sepsis.

Impeding the single-strand annealing pathway of DNA double-strand break repair by withaferin A-mediated FANCA degradation.

FANCA is a key player in the canonical Fanconi anemia (FA) repair pathway. We have recently shown that FANCA also plays an important role in the single-strand annealing sub-pathway (SSA) of DNA double-strand break (DSB) repair by biochemically catalyzing single-strand annealing. Here, we report that a steroidal lactone withaferin A (WA) specifically impedes SSA repair by promoting FANCA downregulation at a sub-micromolar concentration range. We find that WA causes FANCA downregulation post-translationally in a proteasome-dependent manner. This WA-mediated downregulation is achieved through HSP90 inhibition and disruption of the FANCA-HSP90 interaction. WA-mediated FANCA degradation significantly reduces cellular SSA repair, abolishes FANCD2 monoubiquitination, elevates sensitivity to mitomycin C, and results in accumulation of DSBs. Importantly, the WA-induced defect in SSA repair is highly dependent on the absence of FANCA protein and overexpression of exogenous WT-FANCA protein in WA-treated cells significantly complements the repair defect.

Stitching up broken DNA ends by FANCA.

RAD52 rejoins resected broken DNA ends by mediating single-strand annealing. Our recent work elucidates that FANCA, a Fanconi anemia protein, also directly repairs double-strand breaks (DSBs) by catalyzing annealing of single-stranded DNA. FANCA and RAD52 likely play complementary roles to each other to prevent deleterious consequences of DSBs. Abbreviations: DSBs: DNA double-strand breaks; ICL: interstrand crosslink; ssDNA: single-stranded DNA; HR: homologous recombination; SSA: single-strand annealing; MMEJ: microhomology-mediated end joining; NHEJ: non-homologous end joining; GFP: green fluorescence protein.

FANCA Promotes DNA Double-Strand Break Repair by Catalyzing Single-Strand Annealing and Strand Exchange.

FANCA is a component of the Fanconi anemia (FA) core complex that activates DNA interstrand crosslink repair by monoubiquitination of FANCD2. Here, we report that purified FANCA protein catalyzes bidirectional single-strand annealing (SA) and strand exchange (SE) at a level comparable to RAD52, while a disease-causing FANCA mutant, F1263Δ, is defective in both activities. FANCG, which directly interacts with FANCA, dramatically stimulates its SA and SE activities. Alternatively, FANCB, which does not directly interact with FANCA, does not stimulate this activity. Importantly, five other patient-derived FANCA mutants also exhibit deficient SA and SE, suggesting that the biochemical activities of FANCA are relevant to the etiology of FA. A cell-based DNA double-strand break (DSB) repair assay demonstrates that FANCA plays a direct role in the single-strand annealing sub-pathway (SSA) of DSB repair by catalyzing SA, and this role is independent of the canonical FA pathway and RAD52.

Identification of KANSARL as the first cancer predisposition fusion gene specific to the population of European ancestry origin.

Gene fusion is one of the hallmarks of cancer. Recent advances in RNA-seq of cancer transcriptomes have facilitated the discovery of fusion transcripts. In this study, we report identification of a surprisingly large number of fusion transcripts, including six KANSARL (KANSL1-ARL17A) transcripts that resulted from the fusion between the KANSL1 and ARL17A genes using a RNA splicingcode model. Five of these six KANSARL fusion transcripts are novel. By systematic analysis of RNA-seq data of glioblastoma, prostate cancer, lung cancer, breast cancer, and lymphoma from different regions of the World, we have found that KANSARL fusion transcripts were rarely detected in the tumors of individuals from Asia or Africa. In contrast, they exist in 30 - 52% of the tumors from North Americans cancer patients. Analysis of CEPH/Utah Pedigree 1463 has revealed that KANSARL is a familially-inherited fusion gene. Further analysis of RNA-seq datasets of the 1000 Genome Project has indicated that KANSARL fusion gene is specific to 28.9% of the population of European ancestry origin. In summary, we demonstrated that KANSARL is the first cancer predisposition fusion gene associated with genetic backgrounds of European ancestry origin.

Heat shock 70 kDa protein cognate 5 involved in WSSV toleration of Litopenaeus vannamei.

The expression levels of 97 unigenes encoding heat shock proteins of Litopenaeus vannamei was scanned, and ten of them were significantly induced by white spot syndrome virus (WSSV). Among these genes, heat shock 70 kDa protein cognate 5 (LvHSC70-5) was upregulated to the highest extent and subjected to further studies. Subcellular localization assay revealed that LvHSC70-5 was located in the mitochondria. Aside from WSSV infection, unfolded protein response activation and thermal stress could also upregulate LvHSC70-5. Results of reporter gene assay demonstrated that promoter of LvHSC70-5 was activated by L. vannamei heat shock factor protein 1, activating transcription factor 4 and thermal stress. A decrease in the expression of LvHSC70-5 could reduce the aggregation of proteins in hemocytes and the cumulative mortality of WSSV-infected L. vannamei. LvHSC70-5 in L. vannamei hemocytes was upregulated by mild thermal stress. In addition, mild thermal stress, decreased the copy number of WSSV in shrimp muscle and the cumulative mortality of WSSV-infected L. vannamei. Therefore, collecting results suggested that LvHSC70-5 should be involved in WSSV toleration of shrimp L. vannamei.

Maintenance of genome stability by Fanconi anemia proteins.

Persistent dysregulation of the DNA damage response and repair in cells causes genomic instability. The resulting genetic changes permit alterations in growth and proliferation observed in virtually all cancers. However, an unstable genome can serve as a double-edged sword by providing survival advantages in the ability to evade checkpoint signaling, but also creating vulnerabilities through dependency on alternative genomic maintenance factors. The Fanconi anemia pathway comprises an intricate network of DNA damage signaling and repair that are critical for protection against genomic instability. The importance of this pathway is underlined by the severity of the cancer predisposing syndrome Fanconi anemia which can be caused by biallelic mutations in any one of the 21 genes known thus far. This review delineates the roles of the Fanconi anemia pathway and the molecular actions of Fanconi anemia proteins in confronting replicative, oxidative, and mitotic stress.

Identification and functional characterization of a novel Spätzle gene in Litopenaeus vannamei.

Shrimp innate immunity is the first line of resistance against pathogenic bacteria. The Toll-like receptor (TLR)-NF-κB pathway is vital in this immunity process. In this study, a novel Spätzle gene (LvSpz4) of Litopenaeus vannamei was cloned and functionally characterized. The open reading frame of LvSpz4 was 918 bp, which encoded a putative protein with 305 amino acids. LvSpz4 was most expressed in the gills of L. vannamei. This expression was induced by Vibrio alginolyticus or Staphylococcus aureus infection or lipopolysaccharide stimulation. The reporter gene assay showed that LvSpz4 could activate the promoters of Pen4, Drs, AttA, Mtk, and white spot syndrome virus immediate early gene1 in Drosophila Schneider 2 (S2) cells. Knockdown LvSpz4 increased the cumulative mortality of L. vannamei upon V. alginolyticus infection. The unfolded protein response (UPR) induced the expression of LvSpz4 in L. vannamei. Moreover, the promoter of LvSpz4 was activated by L. vannamei X-Box binding protein 1 and activating transcription factor 4 in S2 cells. These results suggested that LvSpz4 was involved in L. vannamei innate immunity and caused the crosstalk between the TLR-NF-κB pathway and UPR.

Characterization of ATPase Activity of P2RX2 Cation Channel.

P2X purinergic receptors are plasma membrane ATP-dependent cation channels that are broadly distributed in the mammalian tissues. P2RX2 is a modulator of auditory sensory hair cell mechanotransduction and plays an important role in hair cell tolerance to noise. In this study, we demonstrate for the first time in vitro and in cochlear neuroepithelium, that P2RX2 possesses the ATPase activity. We observed that the P2RX2 V60L human deafness mutation alters its ability to bind ATP, while the G353R has no effect on ATP binding or hydrolysis. A non-hydrolysable ATP assay using HEK293 cells suggests that ATP hydrolysis plays a significant role in the opening and gating of the P2RX2 ion channel. Moreover, the results of structural modeling of the molecule was in agreement with our experimental observations. These novel findings suggest the intrinsic ATPase activity of P2RX2 and provide molecular insights into the channel opening.

Identification and functional characterization of a glucose regulated protein 94 gene in Litopenaeus vannamei and its responsiveness in WSSV infection.

In the current study, a cDNA of glucose regulated protein 94 (LvGRP94) was cloned from Litopenaeus vannamei. Subcellular localization assay revealed that LvGRP94 expressed in endoplasmic reticulum (ER). And results of reported gene assays demonstrated that the promoter of LvGRP94 was activated by L. vannamei leucine zipper domain transcription factor X-box binding protein 1 (LvXBP1) or heat shock treatment. Furthermore, LvGRP94 was found to highly express in hemocytes as well as in epidermis by real-time RT-PCR. In addition, it was shown that LvGRP94 inhibited by LvXBP1 knocked-down in the hemocytes, was induced by white spot syndrome virus (WSSV) infection, or unfolded protein response (UPR) pathway activation. Importantly, decreasing LvGRP94 reduced the cumulative mortality of WSSV-infected shrimps and WSSV copies in shrimp muscle. These results suggested that LvGRP94 might involve in shrimp UPR pathway as well as WSSV infection.

Down-regulation apoptosis signal-regulating kinase 1 gene reduced the Litopenaeus vannamei hemocyte apoptosis in WSSV infection.

Apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, is crucial in various cellular responses. In the present study, we identified and characterized an ASK1 homolog from Litopenaeus vannamei (LvASK1). The full-length cDNA of LvASK1 was 5400 bp long, with an open reading frame encoding a putative 1420 amino acid protein. LvASK1 was highly expressed in muscle, hemocyte, eyestalk and heart. Real-time RT-PCR analysis showed that the expression of the LvASK1 was upregulated during the white spot syndrome virus (WSSV) challenge. The knocked-down expression of LvASK1 by RNA interference significantly reduced the apoptotic ratio of the hemocytes collected from WSSV-infected L. vannamei. Furthermore, the down-regulation of LvASK1 also decreased the cumulative mortality of WSSV-infected L. vannamei. These results suggested that down-regulation of LvASK1 decreased the apoptotic rate of hemocytes in WSSV-infected shrimp, and that it could contribute to the reduction of cumulative mortality in WSSV-infected L. vannamei.