Major Histocompatibility Complex Dmα/ß Genes and Their Expressional Diversity in Healthy and Diseased Water Buffalo Bubalus bubalis.

BACKGROUND/AIMS: The major histocompatibility complex (MHC) categorized into three (I, II and III) classes elicits the immunogenic response by presenting exogenous peptides to T cells. The MHC-II DM is composed of DMα and DMß, two polypeptide chains, both are encoded by separate MHC genes involved in antigen processing and presentation. Despite the acknowledged role of MHC complex in humans, the literature is silent on the organization and expression of these genes in water buffalo Bubalus bubalis, an agriculturally important animal species. METHODS: We deduced the full-length mRNA sequences of DMα and DMß genes, localized them onto the chromosome 2, assessed their copy number per haploid genome and studied tissue and disease specific expression. RESULTS: The Real Time PCR showed higher expression of both the genes and their seven interacting partners in spleen, gonads and spermatozoa. Significantly, upregulation of DMα and DMß genes and their interacting partners were detected in diseased group of buffaloes as compared to that in healthy ones. CONCLUSION: The upregulation of Bubalus bubalis (BuLA)-DMα and DMß genes and their interacting partners reflect their role in regulating immune responses towards the amelioration of diseases. Work on this line would enhance our understanding on the overall roles of MHC locus, allowing development of possible therapeutic treatment strategies.

First quantitative dosages: Strong correlations between non-5-HT2Rs serotonin receptors on normal human heart valves.

Objectives: Although critical in animal and human development and pathology, a measurement of the quantitative expression of 5-HTR serotonin receptors on animal or human valvular tissues has never been performed. Methods: Quantification of the most frequent 5-HTRs reported as being present in human peripheral tissue was performed using radiolabeled agonists/antagonists. A membrane protein extract from normal human valves (aortic/mitral/tricuspid and some pulmonary) and associated diseased left myocardium, all unusable in clinics, were obtained from the Homograft bank. Results: We analyzed 5-HT1AR/5-HT1B/DR/5-HT2AR/5-HT2BR/5-HT 2CR/5-HT4R/5-HT7R from 28 hearts. We confirmed the presence of tissue and measured the quantitative content for respective proteins in femtomol/mg of protein extracts: for 5-HT2AR (35.9+/-0.7), 5-HT2BR (28.8+/-1.3) but also a newly observed and robust expression for 5-HT4R (38+/-4.2). We identified one, 5-HT1ARs (4.9+/-0.3), and the possible expression, but at a very low level, of previously reported 5-HT1B/DRs (1.3+/-0.5) as well as the new 5-HT7Rs (3.5+/0.1) and 5-HT2CRs (1.2+/-0.1). Interestingly, by using univariate analysis, we were able to observe many correlations between the different 5-HTR levels of expression especially between 5-HT1AR/5-HT1B/DR and also between 5-HT4R/5-HT7R, but none were observed between 5-HT2AR and 5-HT2BR. Using multivariate analyses for a specific 5-HTR level of expression, after adjustment for implantation sites and other 5-HTRs, we found that 5-HT1AR was correlated with 5-HT1B/DR;5-HT4R with 5-HT7R and 5-HT1AR;5-HT2BR with 5-HT2AR only. For 5-HT2C, no correlation was observed. Conclusion: 5-HT2AR/5-HT2BR and 5-HT4R were all observed to have a high and equal level of expression on human valves, but that of 5-HT1AR was more limited. Since these non-5-HT2Rs are coupled with different G-proteins, with specific signaling, theoretically they may control the main 5-HT2R signaling (i.e., PLC/DAG-PKC-ERK/Ras/Src signaling) involved in valvular fibrosis and degeneration.

The Ovarian Development Genes of Bisexual and Parthenogenetic Haemaphysalis longicornis Evaluated by Transcriptomics and Proteomics.

The tick Haemaphysalis longicornis has two reproductive groups: a bisexual group (HLBP) and a parthenogenetic group (HLPP). The comparative molecular regulation of ovarian development in these two groups is unexplored. We conducted transcriptome sequencing and quantitative proteomics on the ovaries of HLBP and HLPP, in different feeding stages, to evaluate the molecular function of genes associated with ovarian development. The ovarian tissues of HLBP and HLPP were divided into three feeding stages (early-fed, partially-fed and engorged). A total of 87,233 genes and 2,833 proteins were annotated in the ovary of H. longicornis in the different feeding stages. The differentially expressed genes (DEGs) of functional pathway analysis indicated that Lysosome, MAPK Signaling Pathway, Phagosome, Regulation of Actin Cytoskeleton, Endocytosis, Apoptosis, Insulin Signaling Pathway, Oxidative Phosphorylation, and Sphingolipid Metabolism were most abundant in the ovary of H. longicornis in the different feeding stages. Comparing the DEGs between HLBP and HLPP revealed that the ABC Transporter, PI3K-Akt Signaling Pathway and cAMP Signaling Pathway were the most enriched and suggested that the functions of signal transduction mechanisms may have changed during ovarian development. The functions of the annotated proteome of ovarian tissues were strongly correlated with the transcriptome annotation results, and these were further validated using quantitative polymerase chain reaction (qPCR). In the HLBP, the expression of cathepsin L, secreted proteins and glycosidase proteins was significantly up-regulated during feeding stages. In the HLPP, the lysozyme, yolk proteins, heat shock protein, glutathione S transferase, myosin and ATP synthase proteins were up-regulated during feeding stages. The significant differences of the gene expression between HLBP and HLPP indicated that variations in the genetic background and molecular function might exist in the two groups. These results provide a foundation for understanding the molecular mechanism and exploring the functions of genes in the ovarian development of different reproductive groups of H. longicornis.

A high throughput assay of lichenase activity with Congo red dye in plants.

BACKGROUND: Since the beginning of the use of reporter proteins for expression analysis, a variety of approaches have been developed and proposed; both qualitative and quantitative. The lack of simple methods for direct observation of gene expression in living organisms makes it necessary to continue to propose new methods. In this work, we consider a method for the quantitative analysis of the expression of thermostable lichenase from Clostridium thermocellum used as a sensitive reporter protein. RESULTS: In this study, we report the design a high throughput fluorometric method for quantification of thermostable lichenase C. thermocellum using Congo red and further experimental verification of its relevance and efficiency in assessment of the functional role of regulatory sequences in the plant cell. CONCLUSIONS: The specific interaction between the dye Congo red and [Formula: see text]-D-glucans formed the background for designing a high-throughput fluorometric assay for quantification of C. thermocellum thermostable lichenase as a reporter protein for plants. This assay (i) makes it possible to precisely measure the amount of reporter protein in a plant sample; (ii) has shown a high sensitivity for quantification of thermostable lichenase; (iii) is more time- and cost-efficient as compared with the Somogyi-Nelson assay; and (iv) is to the least degree dependent on the presence of the tested buffer components as compared with the Somogyi-Nelson assay.

Response of solute transport model parameters to the combination of multiple design parameters and their quantitative expression with hydraulic indicators of FWS-constructed wetlands.

Solute transport models and hydraulic indicators are commonly used to assess the flow pattern of free water surface-constructed wetlands (FWS CWs). However, the relationship between solute transport models and hydraulic performance remains poorly understood. The hybrid model comprised of plug flow with dispersion and continuous stirred tank reactors (PFD + CSTRs) was applied in this study. The variation rules of model parameters, namely the flow ratio of the mixed zone f, volume ratio of the mixed zone z, dispersion number D, and number of mixed tanks N, were obtained by fitting of the normalized tracer data of orthogonal tests. The coefficients of determination (R2) exceeded 0.7 and the correlation coefficient (r) surpassed 0.8. The results demonstrated satisfactory hydraulic performance and purification effect, with high hydraulic and water quality indicators. Water depth was the principal design parameter negatively affecting f and z, whereas the layout of in- and outlet positively influenced D and N. The R2 of the model parameters f, z, and D on most hydraulic indicators were above 0.5. Significantly positive correlations existed between the model parameters f, z, and D and the hydraulic indicators including the short-circuit index φ10, effective volume ratio e, and moment index MI. The quantitative links between model parameters and hydraulic indicators were established. Based on the significant correlations between design parameters, hydraulic indicators, and model parameters, it would be more convenient to evaluate the hydraulic performance of FWS CWs corresponding to specific design parameters. Graphical abstract.

Juvenile myoclonic epilepsy as a spectrum disorder: A focused review.

In consequence of newer research juvenile myoclonic epilepsy (JME) is no longer seen as a homogeneous disease. The causes of the existing variance are only partially known yet. We discuss to what extent the phenotypical spectrum of this polygenetically determined disorder expresses genetically defined endophenotypes, or is due to mere quantitative differences in the expression of the core phenotype. Of the three common seizure types of JME, myoclonic, generalized tonic-clonic and absences, absences also occur independently and are strong candidates for an endophenotype. Focal features may in some patients be seen in clinical seizures or the EEG but rarely in both. They have no morphological correlates. In a system epilepsy, local manifestations are possible, and some are due to reflex mechanisms. Of the four reflex epileptic traits common in JME, photosensitivity and praxis induction appear related to basic mechanisms of the core syndrome, whereas language-induced orofacial reflex myocloni and eye closure sensitivity are also seen in other clinical contexts and therefore seem to represent endophenotypes. Cognitive abnormalities indicating slight frontal lobe dysfunction seem to be ubiquitous in JME and are also seen in unaffected siblings of patients. Cluster B personality disorder is found in 1/3 of patients, representing a more severe expression of the underlying pathology. Treatment response and prognosis seem to be affected by an interplay of the described factors producing the severest end of the JME spectrum. The spectrum appears to be due to an interaction of stronger or weaker expression of the core phenotype with various endophenotypes.

Quantification of expression of dehydrin isoforms in the desiccation tolerant plant Craterostigma plantagineum using specifically designed reference genes.

Craterostigma plantagineum is a desiccation tolerant resurrection plant. Many genes are induced during desiccation. Dehydrins are a group of dehydration-induced genes present in all higher plants. The current study aims at classifying the most abundantly expressed dehydrin genes from vegetative tissues of C. plantagineum and quantifying their expression. To identify variations between dehydrin isoforms at different stages of desiccation and rehydration by RT-qPCR, the target mRNA requires an accurate and reliable normalization. Previously we reported that RNAs from leaves and roots of C. plantagineum are not degraded during desiccation and subsequent rehydration thus allowing the use of RT-qPCR to test the stability of reference genes. The expression stability of eight candidate reference genes was tested in leaves, roots and callus. These genes were ranked according to their stability of gene expression using GeNorm(PLUS) and RefFinder. The most consistently expressed reference genes in each tissue were identified and used to normalize gene expression data. Dehydrin isoforms were divided in three groups based on the expression level during the desiccation process in three different tissues (leaves, roots and callus).