Gel express: a novel frugal method quantifies gene relative expression in conventional RT-PCR

Background Real-time PCR system is a valuable scientific mainstream needed for quantifying specific gene expression. Nevertheless, compared with conventional PCR, the real-time PCR system is extremely expensive and not affordable for limited or mid-budget research laboratories. Here, a novel, doable and low-cost recipe (referred to as gel express) is developed to quantify gene expression using conventional RT-PCR assay. The novelty of the gel express method is based on replacing crossing point (CP) values with integrated density (IntDen) values of PCR amplicon bands in real-time PCR regular mathematical formulas. Results In this work, gene expression profiles of two different rice stress-marker genes (OsCYP94C2a and OsLOX8) were quantified in response to mechanical wounding at different time points (0, 30, 60, and 150 min). In the gel express method, the free software ImageJ was employed to measure integrated density (IntDen) values of PCR amplicon bands in agarose gel images. IntDen values were then used instead of crossing point (CP) values according to the following modified formula: [EIntDen(ref)/EIntDen(target)]sample ÷ [EIntDen(ref)/EIntDen(target)]control. Gene relative expression profiles (dynamic expression pattern) quantified by gel express method in both genes were highly comparable with real-time RT-PCR. R2 values were 0.9976 and 0.9975 in OsCYP94C2a and OsLOX, respectively. PCR amplification efficiency (E) for all studied genes could be calculated depending on IntDen values through experimentally designed calibration curves. PCR amplification efficiencies with all studied genes obtained by gel express were all in the accepted range. For better-visualized PCR amplicons thus detectable biological effects between treatments, the number of PCR cycles applied in gel express method (IntCyc) was experimentally estimated to be 29 cycles. Conclusions Gel express is a novel, cost-effective and feasible recipe for quantifying gene relative expression in conventional RT-PCR. The expression pattern quantified by gel express is highly comparable and fits the expression data revealed by the used real-time PCR system.

The role of the epidermis enhancer element in positive and negative transcriptional regulation of ebony in Drosophila melanogaster

The spatiotemporal regulation of gene expression is essential to ensure robust phenotypic outcomes. Pigmentation patterns in Drosophila are determined by pigments biosynthesized in the developing epidermis and the cis-regulatory elements (CREs) of the genes involved in this process are well-characterized. Here we report that the known primary epidermal enhancer (priEE) is dispensable for the transcriptional activation of ebony (involved in light-colored pigment synthesis) in the developing epidermis of D. melanogaster. The evidence was obtained by introducing an approximately 1 kbp deletion at the priEE by genome editing. The effect of the priEE deletion on pigmentation and on the endogenous expression pattern of a mCherry-fused ebony allele was examined in the abdomen. The expression levels of the mCherry-fused ebony in the priEE-deleted strains were slightly higher than that of the control strain, indicating that the sequences outside the priEE have an ability to drive an expression of this gene in the epidermis. Interestingly, the priEE deletion resulted in a derepression of this gene in the dorsal midline of the abdominal tergites, where dark pigmentation is present in the wild-type individuals. This indicated that the priEE fragment contains a silencer. Furthermore, the endogenous expression pattern of ebony in the two additional strains with partially deleted priEE revealed that the silencer resides within a 351-bp fragment in the 5' portion of the priEE. These results demonstrated that deletion assays combined with reporter assays are highly effective in detecting the presence of positively and negatively regulating sequences within and outside the focal CREs.

An Insight Into Systemic Immune Response in Leishmania donovani Mediated Atypical Cutaneous Leishmaniasis in the New Endemic State of Himachal Pradesh, India

Leishmaniasis continues to afflict known and newer endemic sites despite global efforts towards its control and elimination. In this regard, the emergence of newer endemic sites with unusual disease formats is recognized wherein Leishmania donovani complex classically known to cause visceral disease is demonstrated to cause cutaneous manifestation. In this context, atypical cutaneous leishmaniasis (CL) cases caused by L. donovani genetic variants from the newer endemic state of Himachal Pradesh (HP) in India are beginning to be understood in terms of parasite determinants. The atypical CL manifestation further needs to be explored to define host immune correlates with a possible role in driving the unusual disease progression. In the given study, we performed comprehensive systemic-immune profiling of the atypical CL patients from the study area in HP, India, in comparison with the classical visceral leishmaniasis (VL) patients from the northeast region of India. The systemic immune response was studied using ELISA-based assessment of Th1, Th2, Th17, Treg, and Th22 specific plasma cytokine expression pattern and parasite-specific total serum IgG/IgG subclasses. The specified immune correlates are known to exhibit heterogeneous association with the different infecting parasite species, infection load, and co-lateral host immunopathology in classical CL and VL. In the atypical CL patient group, altered expression of IL-10 emerged as the key finding that could potentially fine-tune the Th1/Th17/Th22 effector cytokine axis towards a localized cutaneous manifestation. A reduced expression of IL-10 along with a high IFN-γ/IL-10 ratio as a readout of effective parasite killing defined atypical cutaneous outcome. In contrast, high circulatory IL-10 levels and a depressed IFN-γ/IL-10 ratio were seen in classical VL patients in line with an ineffective parasite-killing cytokine response. Overall, the study highlights new knowledge on host immune correlates in terms of cytokine expression pattern and IgG subclasses that underline atypical disease manifestation such that L. donovani, a generally visceralizing parasite species cause skin localized cutaneous lesions.

Genome-wide identification and characterization of the CLASP_N gene family in upland cotton (Gossypium hirsutum L.)

Background Cytoplasmic linker–associated proteins (CLASPs) are tubule proteins that can bind to microtubules and participate in regulating the structure and function of microtubules, which significantly affects the development and growth of plants. These proteins have been identified in Arabidopsis; however, little research has been performed in upland cotton. Methods In this study, the whole genome of the CLASP_N family was analyzed to provide theoretical support for the function of this gene family in the development of upland cotton fiber. Bioinformatics was used to analyze the family characteristics of CLASP_N in upland cotton, such as member identification, sequence characteristics, conserved domain structure and coevolutionary relationships. Real-time fluorescent quantitative PCR (qRT-PCR) was used to clarify the expression pattern of the upland cotton CLASP_N gene family in cotton fiber. Results At the genome-wide level, we identified 16 upland cotton CLASP_N genes. A chromosomal localization analysis revealed that these 16 genes were located on 13 chromosomes. The motif results showed that all CLASP_N proteins have the CLASP_N domain. Gene structure analysis showed that the structure and length of exons and introns were consistent in the subgroups. In the evolutionary analysis with other species, the gene family clearly diverged from the other species in the evolutionary process. A promoter sequence analysis showed that this gene family contains a large number of cis-acting elements related to a variety of plant hormones. qRT-PCR was used to clarify the expression pattern of the upland cotton CLASP_N gene family in cotton fiber and leaves, and Gh210800 was found to be highly expressed in the later stages of fiber development. The results of this study provide a foundation for further research on the molecular role of the CLASP_N genes in cotton fiber development.

The GluN2A subunit of the NMDA receptor modulates the rate of functional maturation in parvalbumin-positive interneurons

N-methyl-D-aspartate receptors (NMDARs) are excitatory glutamate-gated ion channels that are expressed throughout the central nervous system. NMDARs mediate calcium entry into cells, and are involved in a host of neurological functions, including neuronal development and maturation. The GluN2A subunit, encoded by the GRIN2A gene, has a slightly delayed expression pattern, with low transcript levels during embryonic development that peak in the early neonatal period. Given its unique expression pattern and ability to speed up the synaptic time course after incorporation into the postsynaptic density compared to other GluN2 subunits, the GluN2A subunit is well positioned to participate in synaptic maturation and circuit refinement. By using Grin2a knockout mice, we show that the loss of GluN2A signaling impacts parvalbumin-positive GABAergic interneuron development in the hippocampal CA1 subfield. Specifically, Grin2a knockout mice have 33% more parvalbumin-positive cells in CA1 compared to wild type controls, with no impact on cholecystokinin-positive cell density. By using immunohistochemical colocalization staining and electrophysiological recordings, we demonstrate that these excess parvalbumin cells do eventually incorporate into the hippocampal network and participate in phasic inhibition, although their presynaptic release probability may be dampened. Moreover, we show that although the morphology of Grin2a knockout parvalbumin-positive cells is unaffected, key measures of intrinsic excitability and action-potential firing properties show age-dependent alterations. Preadolescent (P20-25) parvalbumin-positive cells have an increased input resistance, longer membrane time constant, longer action-potential half-width, a lower current threshold for depolarization-induced block of action-potential firing, and a decrease in peak action-potential firing rate. Each of these electrophysiological measures becomes corrected in adulthood, reaching wild type levels, suggesting a delay of electrophysiological maturation. The circuit and behavioral implications of delayed parvalbumin-positive interneuron maturation are not known; however, we find that neonatal Grin2a knockout mice are more susceptible to lipopolysaccharide and febrile-induced seizures, consistent with a critical role for early GluN2A signaling in neuronal development and maintenance of excitatory-inhibitory balance. These results could provide insights into how loss-of-function GRIN2A human variants can generate an epileptic phenotype.

Dysregulation of complement factor H in juvenile-onset systemic lupus erythematosus patients

Objectives: This study aims to evaluate the expression pattern of factor H in peripheral blood and the frequency of factor H autoantibodies in plasma of juvenile-onset systemic lupus erythematosus (jSLE) patients compared to healthy controls. Patients and methods: Between March 2019 and October 2019, a total of 30 healthy individuals (3 males, 27 females; mean age: 26±7.4 years; range, 18 to 40 years) and 65 jSLE patients (age of onset ≤16 years) (2 males, 63 females; mean age: 23.4±7 years; range, 15 to 38 years) were included. Factor H expression pattern was examined in blood of all subjects using quantitative real-time polymerase chain reaction and the frequency of factor H autoantibodies was estimated in plasma using enzyme-linked immunosorbent assay. Results: Factor H expression was significantly downregulated in jSLE patients compared to healthy controls (p<0.01). A significant underexpression of factor H was observed in jSLE patients with nephritis compared to those without nephritis (p<0.03), while there was no association of factor H expression levels with any of the other clinical and serological features, disease activity or disease damage index of patients. Only 5% of jSLE patients were positive for factor H autoantibodies without any correlations with the clinical data or disease activity of patients. Conclusion: Our study results suggest that factor H expression can be dysregulated in jSLE patients.