Fusion primer and nested integrated PCR (FPNI-PCR): a new high-efficiency strategy for rapid chromosome walking or flanking sequence cloning

<p>Abstract</p> <p>Background</p> <p>The advent of genomics-based technologies has revolutionized many fields of biological enquiry. However, chromosome walking or flanking sequence cloning is still a necessary and important procedure to determining gene structure. Such methods are used to identify T-DNA insertion sites and so are especially relevant for organisms where large T-DNA insertion libraries have been created, such as rice and <it>Arabidopsis</it>. The currently available methods for flanking sequence cloning, including the popular <it>TAIL-PCR </it>technique, are relatively laborious and slow.</p> <p>Results</p> <p>Here, we report a simple and effective fusion primer and nested integrated PCR method (<it>FPNI-PCR</it>) for the identification and cloning of unknown genomic regions flanked known sequences. In brief, a set of universal primers was designed that consisted of various 15-16 base arbitrary degenerate oligonucleotides. These arbitrary degenerate primers were fused to the 3' end of an adaptor oligonucleotide which provided a known sequence without degenerate nucleotides, thereby forming the fusion primers (FPs). These fusion primers are employed in the first step of an integrated nested PCR strategy which defines the overall <it>FPNI-PCR </it>protocol. In order to demonstrate the efficacy of this novel strategy, we have successfully used it to isolate multiple genomic sequences namely, 21 orthologs of genes in various species of Rosaceace, 4 <it>MYB </it>genes of Rosa rugosa, 3 promoters of transcription factors of Petunia hybrida, and 4 flanking sequences of T-DNA insertion sites in transgenic tobacco lines and 6 specific genes from sequenced genome of rice and <it>Arabidopsis</it>.</p> <p>Conclusions</p> <p>The successful amplification of target products through <it>FPNI-PCR </it>verified that this novel strategy is an effective, low cost and simple procedure. Furthermore, <it>FPNI-PCR </it>represents a more sensitive, rapid and accurate technique than the established <it>TAIL-PCR </it>and <it>hiTAIL-PCR </it>procedures.</p

Large‐scale changes in macrobenthic biodiversity driven by mangrove afforestation

1. Large- scale anthropogenic mangroves have been constructed in coastal regions worldwide but our understanding of their ecological effects is limited. In particu-lar, the question of whether and how anthropogenic mangroves influence biodi-versity patterns remains elusive.2. Here, we investigated the influence of large-scale anthropogenic mangroves on biodiversity patterns of mangrove macrobenthos. Specifically, we measure and seek to explain differences in species richness, abundance, assemblage composi-tion and distance-decay effect before and after the construction of anthropo-genic mangroves.3. We surveyed assemblages of gastropod, bivalve and crab species over a wide latitudinal extent (24–28°N) in subtropical China. For each, we calculated species richness, abundance, assemblage composition and distance-decay relationship before and after the construction of anthropogenic mangroves.4. After the large-scale anthropogenic mangroves, we found species richness of gas-tropods, bivalves and crabs increased by 23.81%, 100% and 20%, respectively. The distance-decay effects of gastropods and bivalves decreased by 25% and 91.43%, while that of crabs remained virtually unchanged, which mediated by in-creased dispersal rate of macrobenthos. With mangrove plantation, compositional similarity of crab and bivalve assemblages increased by 28.57% and 38.46%, sug-gesting that large-scale monospecific planting exacerbate biotic homogenization. Altogether, these results indicate that large-scale anthropogenic habitats increase the diversity of mangrove macrobenthos and change taxonomic compositions by reducing distance-decay effects and increasing dispersal rate of macrobenthos.5. Synthesis and applications. We emphasize that afforestation of coastal wetlands can drive major changes in benthonic communities. Monitoring and assessing the ecological effects of the anthropogenic habitats for the presence of functional faunas will be important in determining the future coastal restoration and main-taining economic aquaculture. Quantifying those effects in terms of regional bio-diversity composition will contribute to the management of coastal restoration to be based upon macroevidence rather than a one-sided local perspective.info:eu-repo/semantics/publishedVersio

Efficacy of radiomics model based on the concept of gross tumor volume and clinical target volume in predicting occult lymph node metastasis in non-small cell lung cancer

ObjectiveThis study aimed to establish a predictive model for occult lymph node metastasis (LNM) in patients with clinical stage I-A non-small cell lung cancer (NSCLC) based on contrast-enhanced CT.MethodsA total of 598 patients with stage I–IIA NSCLC from different hospitals were randomized into the training and validation group. The “Radiomics” tool kit of AccuContour software was employed to extract the radiomics features of GTV and CTV from chest-enhanced CT arterial phase pictures. Then, the least absolute shrinkage and selection operator (LASSO) regression analysis was applied to reduce the number of variables and develop GTV, CTV, and GTV+CTV models for predicting occult lymph node metastasis (LNM).ResultsEight optimal radiomics features related to occult LNM were finally identified. The receiver operating characteristic (ROC) curves of the three models showed good predictive effects. The area under the curve (AUC) value of GTV, CTV, and GTV+CTV model in the training group was 0.845, 0.843, and 0.869, respectively. Similarly, the corresponding AUC values in the validation group were 0.821, 0.812, and 0.906. The combined GTV+CTV model exhibited a better predictive performance in the training and validation group by the Delong test (p&lt;0.05). Moreover, the decision curve showed that the combined GTV+CTV predictive model was superior to the GTV or CTV model.ConclusionThe radiomics prediction models based on GTV and CTV can predict occult LNM in patients with clinical stage I–IIA NSCLC preoperatively, and the combined GTV+CTV model is the optimal strategy for clinical application

A transcription factor of the NAC family regulates nitrate-induced legume nodule senescence

17 Pags.- 7 Figs. © 2023 The AuthorsLegumes establish symbioses with rhizobia by forming nitrogen-fixing nodules. Nitrate is amajor environmental factor that affects symbiotic functioning. However, the molecularmechanism of nitrate-induced nodule senescence is poorly understood. Comparative transcriptomic analysis reveals an NAC-type transcription factor inLotus japo-nicus, LjNAC094, that acts as a positive regulator in nitrate-induced nodule senescence.Stable overexpression and mutant lines ofNAC094were constructed and used for phenotypiccharacterization. DNA-affinity purification sequencing was performed to identify NAC094targeting genes and results were confirmed by electrophoretic mobility shift and transactiva-tion assays. Overexpression ofNAC094induces premature nodule senescence. Knocking outNAC094partially relieves nitrate-induced degradation of leghemoglobins and abolishes nodule expres-sion of senescence-associated genes (SAGs) that contain a conserved binding motif forNAC094. Nitrate-triggered metabolic changes in wild-type nodules are largely affected innac094mutant nodules. Induction ofNAC094and its targetingSAGswas almost blocked inthe nitrate-insensitivenlp1,nlp4, andnlp1 nlp4mutants. We conclude that NAC094 functions downstream of NLP1 and NLP4 by regulating nitrate-induced expression ofSAGs. Our study fills in a key gap between nitrate and the execution ofnodule senescence, and provides a potential strategy to improve nitrogen fixation and stresstolerance of legumes.This work was supported by the National Natural Science Foundation of China (32000192, 31870220), the Foundation of Hubei Hongshan Laboratory (2022hszd014), Fundamental Research Funds for the Central Universities (2662020SKPY007), and MCIN/AEI/10.13039/501100011033 (grant PID2020-113985GB-I00). We also thank the BaiChuan fellowship of College of Life Science and Technology, Huazhong Agricultural University, for funding support.Peer reviewe

Single cell-type transcriptome profiling reveals genes that promote nitrogen fixation in the infected and uninfected cells of legume nodules

2 Pags.- 1 Fig. © 2022 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use,distribution and reproduction in any medium, provided the original work is properly cited.Excessive application of nitrogen fertilizers has inevitably resultedin environmental problems. The symbiotic nitrogen fixation (SNF) that occurs in the root nodules of leguminous plants provides asustainable source of reduced nitrogen in agricultural ecosystems. More than 200 genes have been reported to regulate SNF, including rhizobial infection, nodule organogenesis and senescence (Royet al., 2020). Mature nodules consist mainly of twocell types: infected cells (IC) that contain nitrogen-fixing bac-teroids and uninfected cells (UC) that mediate active metabolismand nutrient transport. Although it is well known that SNFrequires functional specialization, the specific genes responsiblefor transcriptional regulation and carbon/nitrogen metabolismand transport in IC and UC remain largely unexplored.Single-cell transcriptomics has emerged as a powerful tech-nique for investigating spatiotemporal patterns of gene expression.This work was supported by the National Natural Science Foundation of China (31870220, 32000192), the China Post-doctoral Science Foundation (2020M680103), Fundamental Research Funds for the Central Universities 2662020SKPY007 and MCIN/AEI/10.13039/501100011033 (grant PID2020-113985GB-I00)Peer reviewe

Reduces distance-decay effect via monospecific mangrove afforestation increase macrobenthic diversity and biomass

Anthropogenic causes are overtaking natural factors to reshape patterns of biodiversity and ecosystem functioning. Mangrove afforestation aimed at reversing losses of mangroves has been conducted worldwide for several decades. However, how afforestation influences the link between ecological processes that shape community diversity and the consequent effects on ecosystem functions such as biomass production is less well known. Here we used data collected before and after mangrove planting to examine the effects of afforestation on molluscan species richness and biomass production by testing the changes in species richness, compositional similarities, distance-decay effects, and metacommunity mechanisms (i.e., species sorting, patch dynamics, mass effects and neutral dynamics) across a regional scale of 480 km (23–27 °N) in southeast Chinese coasts. Additionally, we further detected the impact of landscape configuration caused by different intensities of afforestation on the mollusc community. After the mangrove afforestation, molluscan species richness increased by 17 species for every additional anthropogenic mangrove habitat. The increase in species richness was mediated by reducing distance-decay effects. This result was more pronounced with patch dynamics and species sorting than with neutral dynamics or mass effects. We also found that the relative importance of the four metacommunity mechanisms varies among landscape configurations. Moreover, the increase in species richness via the afforestation indeed leads to a concomitant increase in benthic molluscan biomass production. We highlight the importance of considering the effects of anthropogenic changes on the relationship between biodiversity and ecosystem functioning in a pluralistic way that considers both the multiple ecological processes that shape community diversity and productivity simultaneously in naturally assembled communities. Quantifying those effects in terms of metacommunity mechanisms enables management decisions about coastal restoration to be based upon ecological mechanisms rather than wishful thinking or superficial appearance

Extreme cold events reduce the stability of mangrove soil mollusc community biomass in the context of climate impact

The frequency and intensity of climate extreme events are expected to increase with global warming in the future. Climate extreme events, such as an extreme cold event (ECE) will continue to influence the stability of soil fauna community biomass, since climate changes often cause a shift in community compositions and structures (e.g. biomass). Few studies, however, have addressed the effects of climate extreme events on the stability of soil fauna community biomass. A field investigation was conducted from 2007 to 2010 to assess the influence of an ECE on the biomass stability of the soil mollusc community across four mangrove wetlands (∼450 km) in South China. Distance-decay and time-decay were used to test the spatiotemporal dynamics of the community biomass. Network analysis and null model were performed to detect the importance of competitive interactions in shaping the stability of the soil mollusc community biomass. The ECE reduced the biomass of the soil mollusc community but increased the complexity of the spatiotemporal patterns of the community biomass. The ECE increased divergent temporal succession and spatial segregation in the soil mollusc community biomass, reflecting the spatiotemporal dynamics of the soil mollusc community biomass influenced by the ECE. Importantly, the ECE decreased the biomass stability of the soil mollusc community by an average of 34.17%. An increase in the modularity of an interactive network (by 75%) and a rise in the intensity of species competition were found after the ECE, reflecting that the ECE enhanced the competitive interactions of the soil mollusc community. The changes in the biomass stability of the soil mollusc community potentially impact their ability to provide ecosystem functions and services such as food production and carbon sequestration for humans. In general, these findings provide valuable ecological insights concerning the effects of climate extremes on the stability properties of ecological soil communities, thereby providing potential applications for soil management and predicting climate changes

Thoughts on the Present Situation of the Development of Rural Animal Husbandry and Its Relationship with the Construction of Ecological Agriculture and the Revitalization of Rural Industry

After several rounds of phased ups and downs of development, rural animal husbandry finally presents a shrinking and depressed development situation under the comprehensive action of the failure of transformation and upgrading and the increasing production costs and breeding risks. Actively exploring and guiding the healthy and sound development of rural animal husbandry is not only an important measure to promote the construction of ecological agriculture, but also the main starting point to realize the revitalization of rural industry. This paper analyzes the present situation of the development of rural animal husbandry in recent years, the influence of animal husbandry on the construction of ecological agriculture and the revitalization of rural industry, and puts forward the corresponding measures, in order　to promote the steady and efficient development of animal husbandry in the grand strategy of revitalizing rural industry

DNA methylation-mediated repression of exosomal miR-652-5p expression promotes oesophageal squamous cell carcinoma aggressiveness by targeting PARG and VEGF pathways.

Exosomal microRNAs (miRNAs) have been recently shown to play vital regulatory and communication roles in cancers. In this study, we showed that the expression levels of miR-652-5p in tumour tissues and serum samples of oesophageal squamous cell carcinoma (OSCC) patients were lower compared to non-tumorous tissues and serum samples from healthy subjects, respectively. Decreased expression of miR-652-5p was correlated with TNM stages, lymph node metastasis, and short overall survival (OS). More frequent CpG sites hypermethylation in the upstream of miR-652-5p was found in OSCC tissues compared to adjacent normal tissues. Subsequently, miR-652-5p downregulation promoted the proliferation and metastasis of OSCC, and regulated cell cycle both in cells and in vivo. The dual-luciferase reporter assay confirmed that poly (ADP-ribose) glycohydrolase (PARG) and vascular endothelial growth factor A (VEGFA) were the direct targets of miR-652-5p. Moreover, the delivery of miR-652-5p agomir suppressed tumour growth and metastasis, and inhibited the protein expressions of PARG and VEGFA in nude mice. Taken together, our findings provide novel insight into the molecular mechanism underlying OSCC pathogenesis

Coordination among flower pigments, scents and pollinators in ornamental plants

Abstract Flower color and scent, crucial qualitative characteristics of ornamental plants, display extensive variation. These distinct pigments and scents play a key role in attracting specific pollinators. While previous research primarily delved into the synthetic regulatory mechanisms of individual traits and their respective attraction to insects, recent studies unveil an interconnectedness between flower color and scent through transcriptional regulatory networks. Moreover, evidence suggests that both color and scent actively contribute to insect attraction. This review summarizes the co-regulation and synthesis of pigments and scents, highlighting their pivotal roles in pollinator attraction. The insights provided will serve as valuable references for applications in metabolic engineering, novel variety breeding, and insect and pest detection and management