Expression of Kv4.2 and Kv4.3 potassium channels in human umbilical veins from normal, diabetic and hypertensive pregnancies

Objective: A substantial line of evidence indicates that Kv4.2 and Kv4.3 channels are the major components of rapid transient-outward potassium currents (A-type currents). It is speculated that those currents may be involved in the maintenance of the membrane potential, as well as in the regulation of propagation and frequency of action potentials. However, very little is known about the presence and function of A-type currents in human vascular smooth muscles such as human umbilical vein (HUV). Having in mind its crucial role in the proper fetal oxygenation the aim of the study was to determine whether Kv4.2 and Kv4.3 potassium channels are present in HUV smooth muscle and to investigate potential alterations of their expression during maternal pathological conditions - gestational diabetes mellitus (GDM) and pregnancy-induced hypertension (PIH). Materials and methods: Healthy, diabetic and hypertensive pregnancies were subjects of this investigation. Each group was consisted of 6 HUV samples obtained from 6 normal pregnancies, 6 pregnancies with GDM, and 6 pregnancies with PIH. After pharmacology analysis, immunohistochemistry and Western blot were performed. Results: Immunohistochemistry revealed similar expression pattern of both, Kv4.2 and Kv4.3 subunits, in HUV smooth muscle in all groups of patients. Results obtained by Western blot were in agreement with immunohistochemical staining. The expression of Kv4.2 and Kv4.3 subunits was not significantly different between the groups. Conclusion: Collectively, this is the first study that demonstrated presence of Kv4.2 and Kv4.3 potassium channels in the HUV smooth muscle and their preservation during the course of GDM and PIH. These channels are most likely major components of rapid A-type currents that may be relevant for maternal-fetus blood flow and hence fetal development. Also, they may represent sensors for detecting hemodynamic and/or metabolic changes in the local environment.

Transcriptional regulation of flavonoid biosynthesis in Artemisia annua by AaYABBY5

Artemisia annua is a medicinal plant rich in terpenes and flavonoids with useful biological activities such as antioxidant, anticancer, and antimalarial activities. The transcriptional regulation of flavonoid biosynthesis in A. annua has not been well-studied. In this study, we identified a YABBY family transcription factor, AaYABBY5, as a positive regulator of anthocyanin and total flavonoid contents in A. annua. AaYABBY5 was selected based on its similar expression pattern to the phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and flavonol synthase (FLS) genes. A transient dual-luciferase assay in Nicotiana bethamiana with the AaYABBY5 effector showed a significant increase in the activity of the downstream LUC gene, with reporters AaPAL, AaCHS, AaCHI, and AaUFGT. The yeast one-hybrid system further confirmed the direct activation of these promoters by AaYABBY5. Gene expression analysis of stably transformed AaYABBY5 overexpression, AaYABBY5 antisense, and control plants revealed a significant increase in the expression of AaPAL, AaCHS, AaCHI, AaFLS, AaFSII, AaLDOX, and AaUFGT in AaYABBY5 overexpression plants. Moreover, their total flavonoid content and anthocyanin content were also found to increase. AaYABBY5 antisense plants showed a significant decrease in the expression of flavonoid biosynthetic genes, as well as a decrease in anthocyanin and total flavonoid contents. In addition, phenotypic analysis revealed deep purple-pigmented stems, an increase in the leaf lamina size, and higher trichome densities in AaYABBY5 overexpression plants. Together, these data proved that AaYABBY5 is a positive regulator of flavonoid biosynthesis in A. annua. Our study provides candidate transcription factors for the improvement of flavonoid concentrations in A. annua and can be further extended to elucidate its mechanism of regulating trichome development.

The Functionally Characterization of Putative Genes Involved in the Formation of Mannose in the Aplanospore Cell Wall of Haematococcus pluvialis (Volvocales, Chlorophyta)

Unicellular volvocalean green algal Haematococcus pluvialis, known as astaxanthin rich microalgae, transforms into aplanospore stage from the flagellate stage when exposed to the stress environments. However, the mechanism of the formation of aplanospore cell wall, which hinders the extraction of astaxanthin and the genetic manipulation is still unclear. In this study, the cell wall components under salicylic acid and high light stresses were explored, and cellulose was considered the main component in the flagellates, which changed gradually into mannose in the aplanospore stages. During the period, the genes related to the cellulose and mannose metabolisms were identified based on the RNA-seq data, which presented a similar expression pattern. The positive correlations were observed among these studied genes by Pearson Correlation (PC) analysis, indicating the coordination between pathways of cellulose and mannose metabolism. The study firstly explored the formation mechanism of aplanospore cell wall, which might be of scientific significance in the study of H. pluvialis.

Systematic expression profiling of dprs and DIPs reveals cell surface codes in Drosophila larval peripheral neurons

In complex nervous systems, neurons must identify their correct partners to form synaptic connections. The prevailing model to ensure correct recognition posits that cell surface proteins (CSPs) in individual neurons act as identification tags. Thus, knowing what cells express which CSPs would provide insights into neural development, synaptic connectivity, and nervous system evolution. Here, we investigated expression of dprs and DIPs, two CSP subfamilies belonging to the immunoglobulin superfamily (IgSF), in Drosophila larval motor neurons (MNs), sensory neurons (SNs), peripheral glia and muscles using a collection of GAL4 driver lines. We found that dprs are more broadly expressed than DIPs in MNs and SNs, and each examined neuron expresses a unique combination of dprs and DIPs. Interestingly, many dprs and DIPs are not robustly expressed, but instead, are found in gradient and temporal expression patterns. Hierarchical clustering showed a similar expression pattern of dprs and DIPs in neurons from the same type and with shared synaptic partners, suggesting these CSPs may facilitate synaptic wiring. In addition, the unique expression patterns of dprs and DIPs revealed three uncharacterized MNs - MN23-Ib, MN6-Ib (A2) and MN7-Ib (A2). This study sets the stage for exploring the functions of dprs and DIPs in Drosophila MNs and SNs and provides genetic access to subsets of neurons.

Expression Analysis of NF-κB-Related lncRNAs in Parkinson’s Disease

Parkinson’s disease (PD) has been shown to affect approximately 1% of the persons aged more than 65 years. This multifactorial disorder has been associated with abnormal function of NF-κB signals. In this research, we have evaluated expressions of NF-κB-related long non-coding RNAs in the circulation of PD patients compared with healthy controls. Expression of PACER was lower in total PD patients compared with healthy persons (Ratio of mean expressions (RME)=0.32, P value<0.001). This pattern was also evident among males (RME=0.25, P value<0.001). Expression of DILC was higher in total PD patients (RME=4.07, P value<0.001), and in both sex-based subgroups (RME=3.77, P value=0.01 and RME=4.25, P value<0.001, for females and males, respectively). Similarly, CEBPA was significantly over-expressed in total PD patients (RME=14.76, P value<0.001), and in both sex-based subgroups (RME=12.42, P value<0.001 and RME=15.80, P value<0.001, for females and males, respectively). ATG5 had a similar expression pattern (RME=2.6, P value=1E-08, RME=1.73, P value=0.03 and RME=3.09, P value=1E-07, for total cases, females and males, respectively). H19 was up-regulated in total cases and male cases compared with corresponding controls (RME=2.19, P value<0.001, RME=2.68, P value=0.01, respectively). Finally, HNFA1-AS was down-regulated in all comparisons (RME=0.10, P value=2E-06, RME=0.08, P value<0.001 and RME=0.12, P value<0.001, for total cases, females and males, respectively). Among PD patients, expressions of NKILA and ADINR were robustly correlated with each other (r=0.75, P value=2.40E-10). In addition, expression levels of DICER1-AS were significantly correlated with those of ADINR, PACER and H19 in these patients (r=0.73, P value=1.76E-9; r=0.72, P value=5.15E-09 and r=0.72, P value=3.09E-09, respectively). Correlation analyses among healthy controls revealed robust correlations between CHAST and CEBPA (r=0.84, P value=3.09E-09), NKILA and ADINR (r=0.80, P value=4.24E-12) as well as between DILC and CHAST (r=0.76, P value=1.70E-10). CEBPA had the best parameters among all assessed genes (AUC=0.96, Sensitivity=0.90 and specificity=0.97). DILC and ATG5 were the most appropriate markers after CEBPA with AUC values of 0.82 and 0.80, respectively. Most notably, combination of all genes improved AUC, sensitivity and specificity parameters to 1, 0.97 and 0.99, respectively. Cumulatively, the current study provides evidence for participation of NF-κB-related lncRNAs in the pathoetiology of PD.

Overexpression of four MiTFL1 genes from mango delays the flowering time in transgenic Arabidopsis

Background TERMINAL FLOWER 1 (TFL1) belongs to the phosphatidylethanolamine-binding protein (PEBP) family, which is involved in inflorescence meristem development and represses flowering in several plant species. In the present study, four TFL1 genes were cloned from the mango (Mangifera indica L.) variety ‘SiJiMi’ and named MiTFL1-1, MiTFL1-2, MiTFL1-3 and MiTFL1-4. Results Sequence analysis showed that the encoded MiTFL1 proteins contained a conserved PEBP domain and belonged to the TFL1 group. Expression analysis showed that the MiTFL1 genes were expressed in not only vegetative organs but also reproductive organs and that the expression levels were related to floral development. Overexpression of the four MiTFL1 genes delayed flowering in transgenic Arabidopsis. Additionally, MiTFL1-1 and MiTFL1-3 changed the flower morphology in some transgenic plants. Yeast two-hybrid (Y2H) analysis showed that several stress-related proteins interacted with MiTFL1 proteins. Conclusions The four MiTFL1 genes exhibited a similar expression pattern, and overexpression in Arabidopsis resulted in delayed flowering. Additionally, MiTFL1-1 and MiTFL1-3 overexpression affected floral organ development. Furthermore, the MiTFL1 proteins could interact with bHLH and 14-3-3 proteins. These results indicate that the MiTFL1 genes may play an important role in the flowering process in mango.

Global metabolome profiling of exhaled breath condensates in male smokers with asthma COPD overlap and prediction of the disease

Asthma—chronic obstructive pulmonary disease (COPD) overlap, termed as ACO, is a complex heterogeneous disease characterised by persistent airflow limitation, which manifests features of both asthma and COPD. These patients have a worse prognosis, in terms of more frequent and severe exacerbations, more frequent symptoms, worse quality of life, increased comorbidities and a faster lung function decline. In absence of clear diagnostic or therapeutic guidelines, ACO presents as a challenge to clinicians. The present study aims to investigate whether ACO patients have a distinct exhaled breath condensate (EBC) metabolic profile in comparison to asthma and COPD. A total of 132 age and BMI matched male smokers were recruited in the exploratory phase which consisted of (i) controls = 33 (ii) asthma = 34 (iii) COPD = 30 and (iv) ACO = 35. Using nuclear magnetic resonance (NMR) metabolomics, 8 metabolites (fatty acid, propionate, isopropanol, lactate, acetone, valine, methanol and formate) were identified to be significantly dysregulated in ACO subjects when compared to both, asthma and COPD. The expression of these dysregulated metabolites were further validated in a fresh patient cohort consisting of (i) asthma = 32 (ii) COPD = 32 and (iii) ACO = 40, which exhibited a similar expression pattern. Multivariate receiver operating characteristic (ROC) curves generated using these metabolites provided a robust ACO classification model. The findings were also integrated with previously identified serum metabolites and inflammatory markers to develop a robust predictive model for differentiation of ACO. Our findings suggest that NMR metabolomics of EBC holds potential as a platform to identify robust, non-invasive biomarkers for differentiating ACO from asthma and COPD.

The CaCA superfamily genes in Saccharum: comparative analysis and their functional implications in response to biotic and abiotic stress

Background In plants, Calcium (Ca2+) acts as a universal messenger in various signal transduction pathways, including responses to biotic and abiotic stresses and regulation of cellular and developmental processes. The Ca2+/cation antiporter (CaCA) superfamily proteins play vital roles in the transport of Ca2+ and/or other cations. However, the characteristics of these superfamily members in Saccharum and their evolutionary and functional implications have remained unclear. Results A total of 34 CaCA genes in Saccharum spontaneum, 5 CaCA genes in Saccharum spp. R570, and 14 CaCA genes in Sorghum bicolor were identified and characterized. These genes consisted of the H+/cation exchanger (CAX), cation/Ca2+ exchanger (CCX), EF-hand / CAX (EFCAX), and Mg2+/H+ exchanger (MHX) families, among which the CCX and EFCAX could be classified into three groups while the CAX could be divided into two groups. The exon/intron structures and motif compositions suggested that the members in the same group were highly conserved. Synteny analysis of CaCAs established their orthologous and paralogous relationships among the superfamily in S. spontaneum, R570, and S. bicolor. The results of protein-protein interactions indicated that these CaCA proteins had direct or indirect interactions. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis demonstrated that most members of Saccharum CaCA genes exhibited a similar expression pattern in response to hormonal (abscisic acid, ABA) treatment but played various roles in response to biotic (Sporisorium scitamineum) and abiotic (cold) stresses. Furthermore, ScCAX4, a gene encoding a cytoplasm, plasma membrane and nucleus positioning protein, was isolated from sugarcane. This gene was constitutively expressed in different sugarcane tissues and its expression was only induced at 3 and 6 h time points after ABA treatment, however was inhibited and indued in the whole process under cold and S. scitamineum stresses, respectively. Conclusions This study systematically conducted comparative analyses of CaCA superfamily genes among S. spontaneum, R570, and S. bicolor, delineating their sequence and structure characteristics, classification, evolutionary history, and putative functions. These results not only provided rich gene resources for exploring the molecular mechanism of the CaCA superfamily genes but also offered guidance and reference for research on other gene families in Saccharum.

Involvement of ABA Responsive SVB Genes in the Regulation of Trichome Formation in Arabidopsis

Trichome formation in Arabidopsis is regulated by several key regulators, and plants hormones such as gibberellin, salicylic acid, jasmonic acid and cytokinins have been shown to regulate trichome formation by affecting the transcription or activities of the key regulators. We report here the identification of two abscisic acid (ABA) responsive genes, SMALLER TRICHOMES WITH VARIABLE BRANCHES (SVB) and SVB2 as trichome formation regulator genes in Arabidopsis. The expression levels of SVB and SVB2 were increased in response to ABA treatment, their expression levels were reduced in the ABA biosynthesis mutant aba1-5, and they have similar expression pattern. In addition to the trichome defects reported previously for the svb single mutant, we found that even though the trichome numbers were largely unaffected in both the svb and svb2 single mutants generate by using CRISPR/Cas9 gene editing, the trichome numbers were greatly reduced in the svb svb2 double mutants. On the other hand, trichome numbers were increased in SVB or SVB2 overexpression plants. RT-PCR results show that the expression of the trichome formation key regulator gene ENHANCER OF GLABRA3 (EGL3) was affected in the svb svb2 double mutants. Our results suggest that SVB and SVB2 are ABA responsive genes, and SVB and SVB2 function redundantly to regulate trichome formation in Arabidopsis.

Integrated SMRT Technology with UMI RNA-Seq Reveals the Hub Genes in Stamen Petalody in Camellia oleifera

Male sterility caused by stamen petalody is a key factor for a low fruit set rate and a low yield of Camellia oleifera but can serve as a useful genetic tool because it eliminates the need for artificial emasculation. However, its molecular regulation mechanism still remains unclear. In this study, transcriptome was sequenced and analyzed on two types of bud materials, stamen petalody mutants and normal materials, at six stages of stamen development based on integrated single-molecule real-time (SMRT) technology with unique molecular identifiers (UMI) and RNA-seq technology to identify the hub genes responsible for stamen petalody in C. oleifera. The results show that a large number of alternative splicing events were identified in the transcriptome. A co-expression network analysis of MADSs and all the differentially expressed genes between the mutant stamens and the normal materials showed that four MADS transcription factor genes, CoSEP3.1, CoAGL6, CoSEP3.2, and CoAP3, were predicted to be the hub genes responsible for stamen petalody. Among these four, the expression patterns of CoAGL6 and CoSEP3.2 were consistently high in the mutant samples, but relatively low in the normal samples at six stages, while the patterns of CoSEP3.1 and CoAP3 were initially low in mutants and then were upregulated during development but remained relatively high in the normal materials. Furthermore, the genes with high connectivity to the hub genes showed significantly different expression patterns between the mutant stamens and the normal materials at different stages. qRT-PCR results showed a similar expression pattern of the hub genes in the RNA-seq. These results lay a solid foundation for the directive breeding of C. oleifera varieties and provide references for the genetic breeding of ornamental Camellia varieties.