Altered mRNA and Protein Expression of Monocarboxylate Transporter MCT1 in the Cerebral Cortex and Cerebellum of Prion Protein Knockout Mice

The effect of a cellular prion protein (PrP^c) deficiency on neuroenergetics was primarily analyzed via surveying the expression of genes specifically involved in lactate/pyruvate metabolism, such as monocarboxylate transporters (MCT1, MCT2, MCT4). The aim of the present study was to elucidate a potential involvement of PrP^c in the regulation of energy metabolism in different brain regions. By using quantitative real-time polymerase chain reaction (qRT-PCR), we observed a marked reduction in MCT1 mRNA expression in the cortex of symptomatic Zürich I Prnp^−/− mice, as compared to their wild-type (WT) counterparts. MCT1 downregulation in the cortex was accompanied with significantly decreased expression of the MCT1 functional interplayer, the Na⁺/K⁺ ATPase α2 subunit. Conversely, the MCT1 mRNA level was significantly raised in the cerebellum of Prnp^−/− vs. WT control group, without a substantial change in the Na⁺/K⁺ ATPase α2 subunit expression. To validate the observed mRNA findings, we confirmed the observed change in MCT1 mRNA expression level in the cortex at the protein level. MCT4, highly expressed in tissues that rely on glycolysis as an energy source, exhibited a significant reduction in the hippocampus of Prnp^−/− vs. WT mice. The present study demonstrates that a lack of PrP^c leads to altered MCT1 and MCT4 mRNA/protein expression in different brain regions of Prnp^−/− vs. WT mice. Our findings provide evidence that PrP^c might affect the monocarboxylate intercellular transport, which needs to be confirmed in further studies.     

Validation of Tuba1a as Appropriate Internal Control for Normalization of Gene Expression Analysis during Mouse Lung Development

The expression ratio between the analysed gene and an internal control gene is the most widely used normalization method for quantitative RT-PCR (qRT-PCR) expression analysis. The ideal reference gene for a specific experiment is the one whose expression is not affected by the different experimental conditions tested. In this study, we validate the applicability of five commonly used reference genes during different stages of mouse lung development. The stability of expression of five different reference genes (Tuba1a, Actb Gapdh, Rn18S and Hist4h4) was calculated within five experimental groups using the statistical algorithm of geNorm software. Overall, Tuba1a showed the least variability in expression among the different stages of lung development, while Hist4h4 and Rn18S showed the maximum variability in their expression. Expression analysis of two lung specific markers, surfactant protein C (SftpC) and Clara cell-specific 10 kDA protein (Scgb1a1), normalized to each of the five reference genes tested here, confirmed our results and showed that incorrect reference gene choice can lead to artefacts. Moreover, a combination of two internal controls for normalization of expression analysis during lung development will increase the accuracy and reliability of results.     

Genome-Wide Analysis of the Peroxidase Gene Family and Verification of Lignin Synthesis-Related Genes in Watermelon

Watermelon (Citrullus lanatus) is an important horticultural crop worldwide, but peel cracking caused by peel hardness severely decreases its quality. Lignification is one of the important functions of class III peroxidase (PRX), and its accumulation in the plant cell wall leads to cell thickening and wood hardening. For in-depth physiological and genetical understanding, we studied the relationship between peel hardness and lignin accumulation and the role of PRXs affecting peel lignin biosynthesis using genome-wide bioinformatics analysis. The obtained results showed that lignin accumulation gradually increased to form the peel stone cell structure, and tissue lignification led to peel hardness. A total of 79 ClPRXs (class III) were identified using bioinformatics analysis, which were widely distributed on 11 chromosomes. The constructed phylogenetics indicated that ClPRXs were divided into seven groups and eleven subclasses, and gene members of each group had highly conserved intron structures. Repeated pattern analysis showed that deletion and replication events occurred during the process of ClPRX amplification. However, in the whole-protein sequence alignment analysis, high homology was not observed, although all contained four conserved functional sites. Repeated pattern analysis showed that deletion and replication events occurred during ClPRXs’ amplification process. The prediction of the promoter cis-acting element and qRT-PCR analysis in four tissues (leaf, petiole, stem, and peel) showed different expression patterns for tissue specificity, abiotic stress, and hormone response by providing a genetic basis of the ClPRX gene family involved in a variety of physiological processes in plants. To our knowledge, we for the first time report the key roles of two ClPRXs in watermelon peel lignin synthesis. In conclusion, the extensive data collected in this study can be used for additional functional analysis of ClPRXs in watermelon growth and development and hormone and abiotic stress response.     

Genome-Wide Identification and Characterization of PIN-FORMED (PIN) Gene Family Reveals Role in Developmental and Various Stress Conditions in Triticum aestivum L.

PIN-FORMED (PIN) genes play a crucial role in regulating polar auxin distribution in diverse developmental processes, including tropic responses, embryogenesis, tissue differentiation, and organogenesis. However, the role of PIN-mediated auxin transport in various plant species is poorly understood. Currently, no information is available about this gene family in wheat (Triticum aestivum L.). In the present investigation, we identified the PIN gene family in wheat to understand the evolution of PIN-mediated auxin transport and its role in various developmental processes and under different biotic and abiotic stress conditions. In this study, we performed genome-wide analysis of the PIN gene family in common wheat and identified 44 TaPIN genes through a homology search, further characterizing them to understand their structure, function, and distribution across various tissues. Phylogenetic analyses led to the classification of TaPIN genes into seven different groups, providing evidence of an evolutionary relationship with Arabidopsis thaliana and Oryza sativa. A gene exon/intron structure analysis showed a distinct evolutionary path and predicted the possible gene duplication events. Further, the physical and biochemical properties, conserved motifs, chromosomal, subcellular localization, transmembrane domains, and three-dimensional (3D) structure were also examined using various computational approaches. Cis-elements analysis of TaPIN genes showed that TaPIN promoters consist of phytohormone, plant growth and development, and stress-related cis-elements. In addition, expression profile analysis also revealed that the expression patterns of the TaPIN genes were different in different tissues and developmental stages. Several members of the TaPIN family were induced during biotic and abiotic stress. Moreover, the expression patterns of TaPIN genes were verified by qRT-PCR. The qRT-PCR results also show a similar expression with slight variation. Therefore, the outcome of this study provides basic genomic information on the expression of the TaPIN gene family and will pave the way for dissecting the precise role of TaPINs in plant developmental processes and different stress conditions.     

肠出血性大肠杆菌O157∶H7变种Q蛋白对志贺毒素表达的影响

1株肠出血性大肠杆菌O157∶H7变种EC169菌株,携带stx基因但不表达志贺毒素。通过高效热不对称交错聚合酶链式反应（high-efficiency thermal asymmetric interlaced polymerase chain reaction,hiTAIL-PCR）hiTAILPCR扩增得到EC169 stx1及其上游核苷酸片段并克隆测序,结果表明：EC169 q基因与标准株sakai q基因相比存在6个SNP位点。通过PCR扩增O157∶H7高毒株EC150 q基因全长,并构建表达载体pkk223-q分别转化EC169和低毒株EC157。反转录荧光定量PCR实验结果表明,外源q基因在EC169和EC157重组菌中可高效表达,并引起EC157stx转录水平上调,但EC169重组菌stx转录水平不变。反向乳胶凝集实验结果亦证实EC157重组菌志贺毒素表达量提高,而EC169重组菌志贺毒素表达量不变。Q蛋白变异可能并非EC169志贺毒素不表达的主要原因。