Conserved intronic secondary structures with concealed branch sites regulate alternative splicing of poison exons.

Alternative splicing (AS) generates multiple RNA isoforms and increases the complexities of transcriptomes and proteomes. However, it remains unclear how RNA structures contribute to AS regulation. Here, we systematically search transcriptomes for secondary structures with concealed branch sites (BSs) in the alternatively spliced introns and predict thousands of them from six organisms, of which many are evolutionarily conserved. Intriguingly, a highly conserved stem-loop structure with concealed BSs is found in animal SF3B3 genes and colocalizes with a downstream poison exon (PE). Destabilization of this structure allows increased usage of the BSs and results in enhanced PE inclusion in human and Drosophila cells, leading to decreased expression of SF3B3. This structure is experimentally validated using an in-cell SHAPE-MaP assay. Through RNA interference screens of 28 RNA-binding proteins, we find that this stem-loop structure is sensitive to U2 factors. Furthermore, we find that SF3B3 also facilitates DNA repair and protects genome stability by enhancing interaction between ERCC6/CSB and arrested RNA polymerase II. Importantly, both Drosophila and human cells with the secondary structure mutated by genome editing exhibit altered DNA repair in vivo. This study provides a novel and common mechanism for AS regulation of PEs and reveals a physiological function of SF3B3 in DNA repair.

Minor Spliceosomal 65K/RNPC3 Interacts with ANKRD11 and Mediates HDAC3-Regulated Histone Deacetylation and Transcription.

RNA splicing is crucial in the multilayer regulatory networks for gene expression, making functional interactions with DNA- and other RNA-processing machineries in the nucleus. However, these established couplings are all major spliceosome-related; whether the minor spliceosome is involved remains unclear. Here, through affinity purification using Drosophila lysates, an interaction is identified between the minor spliceosomal 65K/RNPC3 and ANKRD11, a cofactor of histone deacetylase 3 (HDAC3). Using a CRISPR/Cas9 system, Deletion strains are constructed and found that both Dm65KΔ/Δ and Dmankrd11Δ/Δ mutants have reduced histone deacetylation at Lys9 of histone H3 (H3K9) and Lys5 of histone H4 (H4K5) in their heads, exhibiting various neural-related defects. The 65K-ANKRD11 interaction is also conserved in human cells, and the HsANKRD11 middle-uncharacterized domain mediates Hs65K association with HDAC3. Cleavage under targets and tagmentation (CUT&Tag) assays revealed that HsANKRD11 is a bridging factor, which facilitates the synergistic common chromatin-binding of HDAC3 and Hs65K. Knockdown (KD) of HsANKRD11 simultaneously decreased their common binding, resulting in reduced deacetylation of nearby H3K9. Ultimately, this study demonstrates that expression changes of many genes caused by HsANKRD11-KD are due to the decreased common chromatin-binding of HDAC3 and Hs65K and subsequently reduced deacetylation of H3K9, illustrating a novel and conserved coupling mechanism that links the histone deacetylation with minor spliceosome for the regulation of gene expression.

Dickkopf-related protein 1/cytoskeleton-associated protein 4 signaling activation by Helicobacter pylori-induced activator protein-1 promotes gastric tumorigenesis via the PI3K/AKT/mTOR pathway.

BACKGROUND: Gastric cancer (GC) is a common malignant tumor with high incidence and mortality rates globally, especially in East Asian countries. Helicobacter pylori (H. pylori) infection is a significant and independent risk factor for GC. However, its underlying mechanism of action is not fully understood. Dickkopf-related protein (DKK) 1 is a Wnt signaling antagonist, and cytoskeleton-associated protein (CKAP) 4 is a newly identified DKK1 receptor. Recent studies found that the binding of DKK1 to CAKP4 mediated the procancer signaling of DKK1 inde-pendent of Wnt signaling. We hypothesize that H. pylori-induced activation of DKK1/CKAP4 signaling contributes to the initiation and progression of GC. AIM: To investigate the interaction of H. pylori infection, DKK1 and CAKP4 in GC, as well as the underlying molecular mechanisms. METHODS: RNA sequencing was used to identify differentially expressed genes (DEGs) between H. pylori-infected and uninfected primary GC cells. Gain- and loss-of-function experiments were performed to verify the H. pylori-induced upregulation of activator protein-1 (AP-1) in GC cells. A dual-luciferase reporter assay and co-immunoprecipitation were used to determine the binding of AP-1 to the DKK1 promoter and DKK1 to CKAP4. Western blotting and immunohistochemistry detected the expression of DKK1, CKAP4, and phos-phatidylinositol 3-kinase (PI3K) pathway-related proteins in GC cells and tissues. Functional experiments and tumorigenicity in nude mice detected malignant behavior of GC cells in vitro and in vivo. RESULTS: We identified 32 DEGs between primary GC cells with and without H. pylori infection, including JUN, fos-like antigen-1 (FOSL1), and DKK1, and confirmed that the three proteins and CKAP4 were highly expressed in H. pylori-infected GC cells, H. pylori-infected gerbil gastric tissues, and human GC tissues. JUN and FOSL1 form AP-1 to transcriptionally activate DKK1 expression by binding to the DKK1 promoter. Activated DKK1 bound to CKAP4, but not the most common Wnt coreceptor low-density lipoprotein receptor-related protein 5/6, to promote GC cell growth, colony formation, migration, invasion, and xenograft tumor growth in nude mice. All these effects were driven by activation of the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway. Targeting the PI3K signaling pathway by LY294002 inhibited DKK1-mediated CKAP4/PI3K signaling activity and the malignant behavior of GC cells. CONCLUSION: H. pylori induces JUN and FOSL1 expression to form AP-1, which transcriptionally activates DKK1. Binding of DKK1 to KAKP4 contributes to gastric tumorigenesis via the PI3K/AKT/mTOR pathway.

An Alternatively Spliced Variant of METTL3 Mediates Tumor Suppression in Hepatocellular Carcinoma.

Many post-transcriptional mRNA processing steps play crucial roles in tumorigenesis and the progression of cancers, such as N6-methyladenosine (m6A) modification and alternative splicing. Upregulation of methyltransferase-like 3 (METTL3), the catalytic core of the m6A methyltransferase complex, increases m6A levels and results in significant effects on the progression of hepatocellular carcinoma (HCC). However, alternative splicing of METTL3 has not been fully investigated, and the functions of its splice variants remain unclear. Here, we analyzed both our and online transcriptomic data, obtaining 13 splice variants of METTL3 in addition to canonical full-length METTL3-A in HCC cell lines and tissues. Validated by RT-qPCR and Western blotting, we found that METTL3-D, one of the splice variants expressing a truncated METTL3 protein, exhibits higher levels than METTL3-A in normal human livers but lower levels than METTL3-A in HCC tumor tissues and cell lines. Further functional assays demonstrated that METTL3-D expression decreased cellular m6A modification, inhibited the proliferation, migration, and invasion of HCC cells, and was negatively associated with the malignancy of patient tumors, exhibiting functions opposite to those of full-length METTL3-A. This study demonstrates that the METTL3-D splice variant is a tumor suppressor that could potentially be used as a target for HCC therapy.

A review of the pharmacological effects of Arctium lappa (burdock).

Arctium lappa, commonly known as burdock, is being promoted/recommended as a healthy and nutritive food in Chinese societies. Burdock has been used therapeutically in Europe, North America and Asia for hundreds of years. The roots, seeds and leaves of burdock have been investigated in view of its popular uses in traditional Chinese medicine (TCM). In this review, the reported therapeutic effects of the active compounds present in the different botanical parts of burdock are summarized. In the root, the active ingredients have been found to "detoxify" blood in terms of TCM and promote blood circulation to the skin surface, improving the skin quality/texture and curing skin diseases like eczema. Antioxidants and antidiabetic compounds have also been found in the root. In the seeds, some active compounds possess anti-inflammatory effects and potent inhibitory effects on the growth of tumors such as pancreatic carcinoma. In the leaf extract, the active compounds isolated can inhibit the growth of micro-organisms in the oral cavity. The medicinal uses of burdock in treating chronic diseases such as cancers, diabetes and AIDS have been reported. However, it is also essential to be aware of the side effects of burdock including contact dermatitis and other allergic/inflammatory responses that might be evoked by burdock.

Prevalence of epilepsy and alcohol-related risk in Zayul County, Tibet Autonomous Region in China: an initial survey.

OBJECTIVE: The goal of this study was to establish the prevalence rates of epilepsy and alcohol-related risk and the treatment gap in Zayul County, Tibet Autonomous Region, to evaluate the diagnosis and treatment status of these patients. METHODS: A door-to-door epidemiological survey was conducted among all the people living in the towns of Shang Zayul and Xia Zayul. A screening questionnaire for epilepsy was used. Clinical and sociodemographic data were collected from patients confirmed to have epilepsy. An audit was used for patients with epilepsy who had a definite history of alcohol consumption and drinking habit change. RESULTS: A total of 7669 people were investigated. Of these, 180 were confirmed to have epilepsy, which was active in 175. Lifetime prevalence was estimated to be 23.5 per 1000 in this population. None of the patients with active epilepsy had received any reasonable antiepileptic therapy in the week before the survey (treatment gap). Ninety-six percent of patients with epilepsy aged between 20 and 60 drank alcoholic beverages. However, in only 25 (13.9%) patients with epilepsy was the epilepsy related to alcohol; most patients (80.6%) had no definite causes. There were no local health workers who received any formal training on epilepsy before, nor did the local health agencies provide any conventional antiepileptic drugs. CONCLUSION: The prevalence rates of epilepsy in the towns of Shang Zayul and Xia Zayul of the Tibet Autonomous Region were relatively higher than in other areas. The alcohol-related health problems cannot be ignored. This serious situation and the large treatment gap indicate a pressing need for medical, financial and social support in this population.

Loss accounting of environmental pollution within Pearl River Delta region, South China.

Economic development puts pressure on environment through air, water and land degradation, which in turn brings real costs to the economy. Actual economy growth should therefore consider the environmental degradation cost resulted from economic activities. Pearl River Delta (PRD) region as a typical delta area with rapid development in China, is with great significance to understand the loses resulted from environmental pollution. This study conducts an environmental economic accounting within the PRD region from 2011 to 2015 using the environmental degradation cost accounting approach. We identified and calculated the economic, agricultural, industrial and social losses resulting from air, water and waste pollution with different valuation methods, which includes shadow price, replacement costs, market value method, etc. The results showed the total environmental degradation cost ranged from 18.1 to 19.8 billion US$ and the environmental degradation index declined slightly over the years, with significant differences among cities. It implied that the environmental condition of PRD region has been continuously improved over the years, but the capacity of environment control between cities had large differences. Cities in PRD region should therefore take measures tailored to their current situation to optimize their resource endowment and industrial structure, to overcome the conflicts between economic development and environmental protection. For cities with relatively high degradation cost, it is urgent to accelerate the efforts in improving the quality of the environment and ecosystem. For cities with lower degradation cost, it is important to take actions to keep on a sustainable and ecological efficient developing path. MAIN FINDINGS: The total environmental degradation cost of the PRD region is firstly calculated with insights on environmental management.

Sequencing and characterization of leaf transcriptomes of six diploid Nicotiana species.

BACKGROUND: Nicotiana belongs to the Solanaceae family that includes important crops such as tomato, potato, eggplant, and pepper. Nicotiana species are of worldwide economic importance and are important model plants for scientific research. Here we present the comparative analysis of the transcriptomes of six wild diploid Nicotiana species. Wild relatives provide an excellent study system for the analysis of the genetic basis for various traits, especially disease resistance. RESULTS: Whole transcriptome sequencing (RNA-seq) was performed for leaves of six diploid Nicotiana species, i.e. Nicotiana glauca, Nicotiana noctiflora, Nicotiana cordifolia, Nicotiana knightiana, Nicotiana setchellii and Nicotiana tomentosiformis. For each species, 9.0-22.3 Gb high-quality clean data were generated, and 67,073-182,046 transcripts were assembled with lengths greater than 100 bp. Over 90 % of the ORFs in each species had significant similarity with proteins in the NCBI non-redundant protein sequence (NR) database. A total of 2491 homologs were identified and used to construct a phylogenetic tree from the respective transcriptomes in Nicotiana. Bioinformatic analysis identified resistance gene analogs, major transcription factor families, and alkaloid transporter genes linked to plant defense. CONCLUSIONS: This is the first report on the leaf transcriptomes of six wild Nicotiana species by Illumina paired-end sequencing and de novo assembly without a reference genome. These sequence resources hopefully will provide an opportunity for identifying genes involved in plant defense and several important quality traits in wild Nicotiana and will accelerate functional genomic studies and genetic improvement efforts of Nicotiana or other important Solanaceae crops in the future.

Comparative Genomics of Pathogens Causing Brown Spot Disease of Tobacco: Alternaria longipes and Alternaria alternata.

The genus Alternaria is a group of infectious/contagious pathogenic fungi that not only invade a wide range of crops but also induce severe allergic reactions in a part of the human population. In this study, two strains Alternaria longipes cx1 and Alternaria alternata cx2 were isolated from different brown spot lesions on infected tobacco leaves. Their complete genomes were sequenced, de novo assembled, and comparatively analyzed. Phylogenetic analysis revealed that A. longipes cx1 and A. alternata cx2 diverged 3.3 million years ago, indicating a recent event of speciation. Seventeen non-ribosomal peptide synthetase (NRPS) genes and 13 polyketide synthase (PKS) genes in A. longipes cx1 and 13 NRPS genes and 12 PKS genes in A. alternata cx2 were identified in these two strains. Some of these genes were predicted to participate in the synthesis of non-host specific toxins (non-HSTs), such as tenuazonic acid (TeA), alternariol (AOH) and alternariol monomethyl ether (AME). By comparative genome analysis, we uncovered that A. longipes cx1 had more genes putatively involved in pathogen-plant interaction, more carbohydrate-degrading enzymes and more secreted proteins than A. alternata cx2. In summary, our results demonstrate the genomic distinction between A. longipes cx1 and A. altenata cx2. They will not only improve the understanding of the phylogenetic relationship among genus Alternaria, but more importantly provide valuable genomic resources for the investigation of plant-pathogen interaction.

Transcriptomic profile of tobacco in response to Alternaria longipes and Alternaria alternata infections.

Tobacco brown spot caused by Alternaria fungal species is one of the most damaging diseases, and results in significant yield losses. However, little is known about the systematic response of tobacco to this fungal infection. To fill this knowledge gap, de novo assemblies of tobacco leaf transcriptomes were obtained in cultivars V2 and NC89 after the inoculation of either Alternaria longipes (AL) or Alternaria alternata (AA) at three different time points. We studied the gene expression profile of each cultivar-pathogen combination, and identified eight differentially expressed genes shared among all combinations. Gene ontology enrichment analysis of the differentially expressed genes revealed key components during the fungal infection, which included regulation of gene expression (GO:0010468), regulation of RNA metabolic process (GO:0051252), tetrapyrrole binding (GO:0046906), and external encapsulating structure (GO:0030312). Further analyses of the continuously upregulated/downregulated genes and the resistance genes demonstrated that the gene expression profile upon fungal infection was contingent on the specific cultivar and pathogen. In conclusion, this study provides a solid foundation for the investigation of plant-pathogen interaction, and is of great importance for disease prevention and molecular breeding.

Genome of Plant Maca (Lepidium meyenii) Illuminates Genomic Basis for High-Altitude Adaptation in the Central Andes.

Maca (Lepidium meyenii Walp, 2n = 8x = 64), belonging to the Brassicaceae family, is an economic plant cultivated in the central Andes sierra in Peru (4000-4500 m). Considering that the rapid uplift of the central Andes occurred 5-10 million years ago (Ma), an evolutionary question arises regarding how plants such as maca acquire high-altitude adaptation within a short geological period. Here, we report the high-quality genome assembly of maca, in which two closely spaced maca-specific whole-genome duplications (WGDs; ∼6.7 Ma) were identified. Comparative genomic analysis between maca and closely related Brassicaceae species revealed expansions of maca genes and gene families involved in abiotic stress response, hormone signaling pathway, and secondary metabolite biosynthesis via WGDs. The retention and subsequent functional divergence of many duplicated genes may account for the morphological and physiological changes (i.e., small leaf shape and self-fertility) in maca in a high-altitude environment. In addition, some duplicated maca genes were identified with functions in morphological adaptation (i.e., LEAF CURLING RESPONSIVENESS) and abiotic stress response (i.e., GLYCINE-RICH RNA-BINDING PROTEINS and DNA-DAMAGE-REPAIR/TOLERATION 2) under positive selection. Collectively, the maca genome provides useful information to understand the important roles of WGDs in the high-altitude adaptation of plants in the Andes.

SNP-based genetic linkage map of tobacco (Nicotiana tabacum L.) using next-generation RAD sequencing.

BACKGROUND: Tobacco (Nicotiana tabacum L.) is an important model system, which has been widely used in plant physiological studies and it is particularly useful as a bioreactor. Despite its importance, only limited molecular marker resources are available for genome analysis, genetic mapping and breeding. Restriction-site associated DNA sequencing (RAD-seq) is a powerful new method for targeted sequencing across the genomes of many individuals. This approach has broad potential for genetic analysis through linkage mapping. RESULTS: We constructed a RAD library using genomic DNA from a BC1 backcross population. Sequencing of 196 individuals was performed on an Illumina HiSeq 2500. Two linkage maps were constructed, one with a reference genome and another, termed as de novo identification of single nucleotide polymorphism (SNP) by RAD-seq, without a reference genome. Overall, 4138 and 2162 SNP markers with a total length of 1944.74 and 2000.9 cM were mapped to 24 linkage groups in the genetic maps based on reference genome and without reference, respectively. CONCLUSIONS: Using two different SNP discovery methods based on next generation RAD sequencing technology, we have respectively mapped 2162 and 4318 SNPs in our backcross population. This study gives an excellent example for high density linkage map construction, irrespective of genome sequence availability, and provides saturated information for downstream genetic investigations such as quantitative trait locus analyses or genomic selection (e.g. bioreactor suitable cultivars).

Total arthroscopic internal drainage technique for the treatment of popliteal cyst / 中国骨伤

OBJECTIVE@#To compare the clinical effects of total arthroscopic internal drainage and arthroscopic combined with posterior small incision in the treatment of popliteal cyst.@*METHODS@#From January 2015 to January 2017, 60 patients with popliteal cyst were treated, including 29 males and 31 females, aged 30 to 65(47.8±2.5) years old, with a course of disease (8.5±4.2) months. Among them, 30 cases received total arthroscopic internal drainage for popliteal fossa cyst(total arthroscopic group), 30 cases received arthroscopic combined with posterior small incision for popliteal fossa cyst(arthroscopic combined with small incision group). The operation time, intraoperative bleeding volume, incision length, Rauschning and Lindgren grade 0 recovery rate and Lysholm score were compared between the two groups.@*RESULTS@#Twenty-nine patients in total arthroscopy group were followed up, and 28 patients in arthroscopy combined with small incision group were followed up for 8 to 20(12.8±2.1) months. Operation time: total arthroscopic group(45.32±5.71) min, arthroscopic combined small incision group (44.56±3.85) min; Rauschning and Lindgren grade 0 recovery: 23 cases in total arthroscopic group, 22 cases in arthroscopic combined small incision group; postoperative Lysholm score: total arthroscopic group 84.5±11.2, arthroscopic combined small incision group 83.2±12.7; there was no significant difference between the two groups(>0.05). Intraoperative bleeding volume: total arthroscopic group(5.32±1.25) ml, arthroscopic combined small incision group(20.75±8.18) ml; incision length: total arthroscopic group (1.51±0.34) cm, arthroscopic combined small incision group (7.34±0.75) cm; the difference between the two groups was significant(<0.05). At the last follow-up, the knee joint was examined by magnetic resonance imaging, and no recurrence of cyst was found.@*CONCLUSIONS@#Total arthroscopic internal drainage and arthroscopic combined with posterior small incision technique for popliteal fossa cyst with intra-articular lesions have the same clinical effect, but less trauma and faster recovery.

Clinical study on the association of ocular dominance with accommodation in myopia adult / 中华实验眼科杂志

Background Dominant eye is one of the functional asymmetric organ,and the dfference between dominant eye and undominant eye is a researching hotspot.But the study about accommodation in adult myopia is less.Objective This study was to determine the association between ocular dominances and accommodative factors in the subjects with adult myopia.Methods This study used prospective descriptive research method.Thirty-five subjects aged from 18 to 35 years with the myopia ranged from-2.00 D to-10.00 D and anisometropia less than 1.5 D,BCVA≥ 1.0 were recruited consecutively in this study.Ocular dominance was determined using the hole-inthe-card test and thumb test.Refractive error was measured with objective and subjective optometry,and amplitude of accommodation was measured by push-up test.Fusion cross cylinder(FCC) was used to measure the accommodative lag,and flipper test was applied to determine the accommodative facility.Oral informed consent was obtained from each subject before any relevant examination.Results No significant differences were found in the amplitude of accommodation (D),accommodative facility (cpm) and accommodative lag (D) between the dominant eye and undominant eye (accommodative amplitude:9.69 D±2.30 D vs.9.60 D±2.37 D,P =0.294 ;accommodative facility: 11.08 D±4.20 D vs.10.63 D± 4.60 D,P=0.260;accommodative lag:P=0.141).In the patients with the right eyes as dominance eyes,the accommodative amplitude of both eyes were (9.48±2.29) cpm and (9.33 ± 2.49) cpm,and accommodative facility were (10.50 ± 4.70) cpm and (9.99 ± 4.90) cpm.There were no significant differences between the right and left eyes in the accommodative amplitude,accommodative facility and accommodative lag (P =0.319,0.116,0.590).In the patients with the left eyes as dominant eyes,the accommodative amplitude of both eyes were (9.91±2.35)D and (9.88±2.26) D,and accommodative facility were (10.70±3.77)cpm and (11.25 ±4.27) cpm.No significant differences were seen between the right eyes and left eyes in the accommodative amplitude,accommodative facility and accommodative lag (P =0.749,0.295,0.238).Conclusions The amplitude of accommodation of the dominant eye is not significantly enhanced,and less accommodative lag and better accommodative facility also are found in the demonstrate eye in myopia adults with low anisometropia.

Quantitative Determination of Tetrandrine in "Zhitong Ruanjiaonang" -An Analgesic Soft Gelatine Capsule Preparation / 中草药

A method for the quantitative determination of tetrandrine in "Zhitong Ruanjiaonang"wasdeveloped by means of TLC densitometry. The method is easy, rapid, accurate, and repro-ducible . The average recovery was 99.98%.

Preparation of Cuiru Oral Liquid / 中草药

Cuiru oral liquid (COL ) for the promotion of lactation is prepared from an aqueous extract of Angelica sinensis, Astragalus membranaceus (Fisch.) Bge., Rehmbnnia glutinosa Libosch.,et al. Its formulatiou and processing were briefly desctibed, and the quality standard and stability of the finished product were studied. Results showed that the formu lation processing and stability of COL were suitable for clinical trial. Moreover, its pharmacology was briefly described.