Structure and repair of replication-coupled DNA breaks.

Using CRISPR/Cas9 nicking enzymes, we examine the interaction between the replication machinery and single strand breaks, one of the most common forms of endogenous DNA damage. We show that replication fork collapse at leading strand nicks generates resected single-ended double-strand breaks (seDSBs) that are repaired by homologous recombination (HR). If these seDSBs are not promptly repaired, arrival of adjacent forks creates double ended DSBs (deDSBs), which could drive genomic scarring in HR-deficient cancers. deDSBs can also be generated directly when the replication fork bypasses lagging strand nicks. Unlike deDSBs produced independently of replication, end-resection at nick-induced se/deDSBs is BRCA1-independent. Nevertheless, BRCA1 antagonizes 53BP1 suppression of RAD51 filament formation. These results highlight unique mechanisms that maintain replication fork stability.

C-to-G editing generates double-strand breaks causing deletion, transversion and translocation.

Base editors (BEs) introduce base substitutions without double-strand DNA cleavage. Besides precise substitutions, BEs generate low-frequency 'stochastic' byproducts through unclear mechanisms. Here, we performed in-depth outcome profiling and genetic dissection, revealing that C-to-G BEs (CGBEs) generate substantial amounts of intermediate double-strand breaks (DSBs), which are at the centre of several byproducts. Imperfect DSB end-joining leads to small deletions via end-resection, templated insertions or aberrant transversions during end fill-in. Chromosomal translocations were detected between the editing target and off-targets of Cas9/deaminase origin. Genetic screenings of DNA repair factors disclosed a central role of abasic site processing in DSB formation. Shielding of abasic sites by the suicide enzyme HMCES reduced CGBE-initiated DSBs, providing an effective way to minimize DSB-triggered events without affecting substitutions. This work demonstrates that CGBEs can initiate deleterious intermediate DSBs and therefore require careful consideration for therapeutic applications, and that HMCES-aided CGBEs hold promise as safer tools.

DNA Damage Atlas: an atlas of DNA damage and repair.

DNA damage and its improper repair are the major source of genomic alterations responsible for many human diseases, particularly cancer. To aid researchers in understanding the underlying mechanisms of genome instability, a number of genome-wide profiling approaches have been developed to monitor DNA damage and repair events. The rapid accumulation of published datasets underscores the critical necessity of a comprehensive database to curate sequencing data on DNA damage and repair intermediates. Here, we present DNA Damage Atlas (DDA, http://www.bioinformaticspa.com/DDA/), the first large-scale repository of DNA damage and repair information. Currently, DDA comprises 6,030 samples from 262 datasets by 59 technologies, covering 16 species, 10 types of damage and 135 treatments. Data collected in DDA was processed through a standardized workflow, including quality checks, hotspots identification and a series of feature characterization for the hotspots. Notably, DDA encompasses analyses of highly repetitive regions, ribosomal DNA and telomere. DDA offers a user-friendly interface that facilitates browsing, searching, genome browser visualization, hotspots comparison and data downloading, enabling convenient and thorough exploration for datasets of interest. In summary, DDA will stand as a valuable resource for research in genome instability and its association with diseases.

Behavioural responses of zebrafish with sound stimuli in microfluidics.

Neuronal activities of the human brain responsible for cognitive features have been theorized through several animal models that exhibited various complementary spatial learning modes by generating a flexible repertoire of behavioral strategies. However, for such studies associated with a neurodegenerative disease, which can be further manipulated to provide therapeutic strategies, the animal models employed in their developmental stages have been preferred over the adult ones. This pilot work was incepted to underscore the spatial memory capabilities that strengthened the intricate mechanism of memory acquisition potential in one of the low-order evolutionarily conserved species, such as zebrafish larvae. Initially, a reliable and more easily reproducible microfluidic platform integrating simple and intricate paths was designed to learn and test the spatial information in zebrafish larvae of 4-6 d.p.f. under non-invasive acoustic stimuli. Further, to acquire spatial information as the representation of spatial memory formation in zebrafish larvae, the acoustic startle responses were evaluated by quantifying various dynamic behaviors under distinct operating parameters. After significant conditioning sessions, the spatial memory was tested by employing variable 'freezing'. By the end of the 30 min-long test session, 6 d.p.f. larvae were found to exhibit the highest value of freezing of approximately 43% and 20% in the short and long paths, respectively. Even though a substantial rate of memory loss was observed, it can be envisaged to serve several behavioral strategies that process the dynamic cognitive memory among distinct spatiotemporal environments. Further, the proposed behavioral paradigm had the advantage of being more adaptable and reliably replicable by other researchers. As a consequence, different hypotheses can be readily tested to generate more reproducible findings towards distinct neurobehavioral characteristics. Therefore, the proposed paradigm for the consolidation of spatial memory based on the non-invasive spatial avoidance strategies could provide an enduring framework of reference for behavioral studies using zebrafish larvae.

LncRNA CCTT-mediated RNA-DNA and RNA-protein interactions facilitate the recruitment of CENP-C to centromeric DNA during kinetochore assembly.

Kinetochore assembly on centromeres is central for chromosome segregation, and defects in this process cause mitotic errors and aneuploidy. Besides the well-established protein network, emerging evidence suggests the involvement of regulatory RNA in kinetochore assembly; however, it has remained elusive about the identity of such RNA, let alone its mechanism of action in this critical process. Here, we report CCTT, a previously uncharacterized long non-coding RNA (lncRNA) transcribed from the arm of human chromosome 17, which plays a vital role in kinetochore assembly. We show that CCTT highly localizes to all centromeres via the formation of RNA-DNA triplex and specifically interacts with CENP-C to help engage this blueprint protein in centromeres, and consequently, CCTT loss triggers extensive mitotic errors and aneuploidy. These findings uncover a non-centromere-derived lncRNA that recruits CENP-C to centromeres and shed critical lights on the function of centromeric DNA sequences as anchor points for kinetochore assembly.

Understanding New Yorkers' Everyday Life Information Seeking Behavior in the Context of Coping with COVID-19.

This poster presents preliminary findings of a study which examines New Yorkers' everyday life information seeking (ELIS) behavior in the context of coping with COVID-19. Based on semi-structured interviews with 10 New Yorkers, this study identified 15 major categories of everyday life information needs, and developed a typology of New Yorkers' preferred ELIS sources. These typologies show New Yorkers' specific ELIS behavior during the pandemic, such as actively seeking information regarding grocery shopping and delivery, health safety guidelines, and current events. Social media became a popular source for immediate, local, and relevant information in New Yorkers' daily lives. These findings could form a knowledge base to inform the development of information services to assist residents of large urban areas in coping with public health crises.

Active retrotransposons help maintain pericentromeric heterochromatin required for faithful cell division.

Retrotransposons are populated in vertebrate genomes, and when active, are thought to cause genome instability with potential benefit to genome evolution. Retrotransposon-derived RNAs are also known to give rise to small endo-siRNAs to help maintain heterochromatin at their sites of transcription; however, as not all heterochromatic regions are equally active in transcription, it remains unclear how heterochromatin is maintained across the genome. Here, we address these problems by defining the origins of repeat-derived RNAs and their specific chromatin locations in Drosophila S2 cells. We demonstrate that repeat RNAs are predominantly derived from active gypsy elements and processed by Dcr-2 into small RNAs to help maintain pericentromeric heterochromatin. We also show in cultured S2 cells that synthetic repeat-derived endo-siRNA mimics are sufficient to rescue Dcr-2-deficiency-induced defects in heterochromatin formation in interphase and chromosome segregation during mitosis, demonstrating that active retrotransposons are required for stable genetic inheritance.

LncRNA HRCEG, regulated by HDAC1, inhibits cells proliferation and epithelial-mesenchymal-transition in gastric cancer.

Recently, a number of long noncoding RNAs (lncRNAs) have been reported to play significant roles in human tumorigenesis. However, only few gastric cancer related lncRNAs have been well characterized. Here, we identified one lncRNA HRCEG, whose expression was decreased in the gastric cancer tissues compared with adjacent normal tissues. Overexpression of HRCEG significantly promoted cell apoptosis and inhibited cell proliferation. Importantly, we demonstrated that HRCEG levels inversely correlated with EMT process and HRCEG was regulated by the histone deacetylase 1 (HDAC1) in gastric cancer. These findings suggest that HRCEG might be regulated by HDAC1 to inhibit gastric cancer progress and metastatic capability via EMT pathway.

Development of a novel DNA delivery system based on rice bran polysaccharide-Fe(III) complexes.

Metal complexes, as a type of potential non-virus gene carriers, have gained much attention due to their properties of high charge density and unique three-dimensional structure. This study investigated the potential of polyethyleneimine (PEI)-modified rice bran polysaccharide-Fe(III) complex (PEI-PI) as a safe gene delivery system and explored the effect of Fe(III) on the efficiency of gene transfection mediated by PEI modified rice bran polysaccharide (PEI-P) and PEI-PI. Gel retardation assay was used to study the DNA binding and protection capability, MTT assay was performed to evaluate the biocompatibility, and PEI-PI complex-mediated EGFP gene transfection was studied in vitro. Results showed the PEI-PI could induce DNA condensation and protect DNA from degradation by DNase I at a low weight ratio (vector/DNA) of 2. At the same weight ratio, PEI exhibited the strongest DNA binding capability but PEI-PI exhibited the highest gene transfection efficiency among all carrier systems. Compared with the PEI-P + Fe(III) system, PEI-PI not only had a more significant capability to condense DNA but also presented higher gene transfection efficiency. Moreover, PEI-PI exhibited no obvious cytotoxicity to cells. This work provides a strategy for the design and development of gene vectors based on PI complexes.

TAT-functionalized PEI-grafting rice bran polysaccharides for safe and efficient gene delivery.

Polysaccharides are considered to be promising candidates for non-viral gene delivery because of their molecular diversity, which can be modified to fine-tune their physicochemical properties. In this work, transcriptional activator protein (TAT) functionalized PEI grafted polysaccharide polymer (PRBP) was prepared by using rice bran polysaccharide as the starting material, and characterized by various methods. The potential of TAT functionalized PRBP (PRBP-TAT) as gene vector was studied in vitro, including DNA loading capacity, DNA protection ability and biocompatibility. The cell uptake and transfection efficiency of the PRBP-TAT/pDNA polyplexes were studied. The results showed that PRBP-TAT could completely condense DNA at N/P 2. The PRBP-TAT/pDNA polyplexes could protect DNA from degrading by DNase and were efficiently internalized by cells. Biocompatibility result showed that PRBP-TAT had no significant cytotoxicity and effect on cell proliferation. At low N/P ratios of 1-3.5, PRBP-TAT showed higher transfection efficiency than PEI30k and PEI30k-grafted rice bran polysaccharide. PRBP-TAT and PEI showed the highest transfection efficiency of 42.8% and 28.1% when pDNA is 2 µg and N/P ratio is 1.5, respectively, while PRBP showed the highest transfection efficiency of 37.3% at N/P 2.5. These results indicate that PTA is a promising candidate vector for safe and efficient gene delivery.

Development of a safe and efficient gene delivery system based on a biodegradable tannic acid backbone.

Finding a safe and efficient gene delivery vector is a major international challenge facing the development of gene therapy. Tannic acid (TA) is a natural cross-linker owing to its hydroxyl and carboxyl groups that can interact with biopolymers for different biomaterial design. In this work, three polyethyleneimine-modified TA polymers were prepared, and the polymers were characterized by FTIR, UV-vis, elemental analysis and 1H NMR. The potential of PTAs as gene vector was studied in vitro, including DNA loading capacity, DNA protection ability and biocompatibility. In addition, the particle size, zeta potential, DNA encapsulation efficiency, cell uptake and transfection efficiency of the PTA-pDNA polyplexes were also studied. The results showed that PTA2k and PTA30k could completely condense DNA at N/P of 2, and PTA600 could only completely condense DNA at N/P of 50. The PTA/pDNA polyplexes could protect DNA from degrading by DNA enzymes and could be efficiently uptaked by cells. Biocompatibility assay showed that PTA had no significant cytotoxicity and effect on cell proliferation compared to PEI. At low N/P ratios of 1-4, PTA showed higher transfection efficiency than PEI, and the transfection efficiency increased with the increase of PEI molecular weight in PTA. At N/P of 3, PTA30k showed the highest transfection efficiency of 23.8%, while PEI30k showed only 6.7%. These results indicate that PTA is a promising candidate vector for safe and efficient gene delivery.

Safe and efficient gene delivery based on rice bran polysaccharide.

Polysaccharides are capable of being modified by polycations to adjust their physical and chemical properties, which accordingly are considered as potential candidate materials for safe and efficient gene delivery. Here, we extracted and purified polysaccharides from rice bran, and their physicochemical properties were determined by various methods. Polyethyleneimine (PEI) modified rice bran polysaccharide (PRBP) was prepared by grafting RBP with low molecular weight PEI and the preparation was determined by FTIR. The potential of PRBP as a gene vector was systematically evaluated in vitro. The results show that PRBP can compact DNA and form PRBP/DNA polylexes with a particle size of 50-100 nm. The PRBP/DNA polylexes can protect DNA degradation from DNase I efficiently. Compared with PEI, higher transfection efficiency was achieved by the PRBP. At weight ratio of 3, the highest efficiency of gene transfection mediated by PRBP-2000 was obtained, which was 37.5% and significantly higher than PEI and commercial reagents (calcium phosphate cell transfection kit) and was closed to lipo6000. Furthermore, according to MTT results, the cytotoxicity of PRBP is much lower than that of PEI, especially for PEI2000. We hope these results will provide new strategy for rice bran polysaccharides development and application as biomaterials.

Rice bran polysaccharide-metal complexes showed safe antioxidant activity in vitro.

The effect on the intracellular reactive oxygen species (ROS) generation, and the antioxidant and cytotoxicity properties of rice bran polysaccharides (RBP) and RBP-metal complexes RBP-Fe(III), RBP-Cu, RBP-Zn and RBP-Ca, were evaluated using atomic absorption spectroscopy (AAS), scavenging activity assays, cell viability assay and fluorescence microscopy. The RBP-metal complexes were prepared using the hydrothermal method. The RBP-Fe(III) complexes were found to be potent scavengers for superoxide (O2-) free radicals. The RBP alone and RBP-Ca complex showed high scavenging activity for 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals. In addition, the RBP-Fe(III) complex also showed good biocompatibility and lowered the intracellular ROS levels, while RBP alone, RBP-Zn and RBP-Ca complexes were observed to increase the intracellular ROS level. Our findings suggest that among the tested RBP-metal complexes, RBP-Fe(III) complex is a strong candidate as an antioxidant therapeutic.

Researchers' participation in and motivations for engaging with research information management systems.

RESEARCHERS' PARTICIPATION IN ONLINE RIMSS: This article examined how researchers participated in research information management systems (RIMSs), their motivations for participation, and their priorities for those motivations. Profile maintenance, question-answering, and endorsement activities were used to define three cumulatively increasing levels of participation: Readers, Record Managers, and Community Members. Junior researchers were more engaged in RIMSs than were senior researchers. Postdocs had significantly higher odds of endorsing other researchers for skills and being categorized as Community Members than did full and associate professors. Assistant professors were significantly more likely to be Record Managers than were members of any other seniority categories. Finally, researchers from the life sciences showed a significantly higher propensity for being Community Members than Readers and Record Managers when compared with researchers from engineering and the physical sciences, respectively. RESEARCHERS' MOTIVATIONS TO PARTICIPATE IN RIMSS: When performing activities, researchers were motivated by the desire to share scholarship, feel competent, experience a sense of enjoyment, improve their status, and build ties with other members of the community. Moreover, when researchers performed activities that directly benefited other members of a RIMS, they assigned higher priorities to intrinsic motivations, such as perceived self-efficacy, enjoyment, and building community ties. Researchers at different stages of their academic careers and disciplines ranked some of the motivations for engaging with RIMSs differently. The general model of research participation in RIMSs; the relationships among RIMS activities; the motivation scales for activities; and the activity, seniority, and discipline-specific priorities for the motivations developed by this study provide the foundation for a framework for researcher participation in RIMSs. This framework can be used by RIMSs and institutional repositories to develop tools and design mechanisms to increase researchers' engagement in RIMSs.

Mesenchymal Stem Cells Promote Hepatocarcinogenesis via lncRNA-MUF Interaction with ANXA2 and miR-34a.

Accumulating evidence suggests that cancer-associated mesenchymal stem cells (MSC) contribute to the development and metastasis of hepatocellular carcinoma (HCC). Aberrant expression of long noncoding RNAs (lncRNA) has been associated with these processes but cellular mechanisms are obscure. In this study, we report that HCC-associated mesenchymal stem cells (HCC-MSC) promote epithelial-mesenchymal transition (EMT) and liver tumorigenesis. We identified a novel lncRNA that we termed lncRNA-MUF (MSC-upregulated factor) that is highly expressed in HCC tissues and correlated with poor prognosis. Depleting lncRNA-MUF in HCC cells repressed EMT and inhibited their tumorigenic potential. Conversely, lncRNA-MUF overexpression accelerated EMT and malignant capacity. Mechanistic investigations showed that lncRNA-MUF bound Annexin A2 (ANXA2) and activated Wnt/ß-catenin signaling and EMT. Furthermore, lncRNA-MUF acted as a competing endogenous RNA for miR-34a, leading to Snail1 upregulation and EMT activation. Collectively, our findings establish a lncRNA-mediated process in MSC that facilitates hepatocarcinogenesis, with potential implications for therapeutic targeting. Cancer Res; 77(23); 6704-16. ©2017 AACR.

[Study on the correlation between hypertension and the indexes of vascular endothelial function among people living in the community].

OBJECTIVE: To study the features of hypertension and vessel endothelium functional parameter in people living at the community level as well as the risk factors of hypertension. Differences of angiotensin II (Ang II ), prostacyclin (PGI2) and nitric oxide (NO) among normal group and three hypertension groups were also studied. METHODS: By cluster sampling, 1134 adult Han people were selected from the residential communities. Medical history was documented and measurements of body height, body weight, waist circumference, hip circumference and blood pressure were performed. Serum NO levels were determined by cadmium reduction method while plasma Ang II and PGI2 concentration were determined by radioimmunoassay. SPSS 13.0 was used for data analysis. RESULTS: The total ratio of hypertension from people living at the community was 44.5%, with the standardized prevalence of hypertension as 15.3%. With the increase of age, the prevalence of hypertension also increased. Overweight and obesity seemed to be independent risk factors for hypertension. History of smoking and drinking and gender did not enter the logistic equation for hypertension. The amount of plasma Ang II concentration of the three hypertension groups was significantly lower than that in the normal group while the lowest group appeared to from the one that hypertension was under control. The NO and PGI2 levels of the two groups whose hypertension had been known were significantly higher than in the normal group while the difference between the group whose hypertension had not been measured and the normal group was not found. CONCLUSION: The prevalence of hypertension had been increasing. Control of body weight seemed to be a useful way for prevention of hypertension. We assumed that the negative feedback regulation of renin-angiotonin-aldosterone system in hypertension patient still existed which called for the research on the mechanism of hypertension.