Sequence Characterization and Spatiotemporal Expression Patterns of PbS

Many flowering plants exhibit an important intraspecific reproductive barrier phenomenon, that is, self-incompatibility (SI), in which S-RNase genes play a significant role. To clarify the specific function of S-RNase genes in Chinese pears, the full length cDNA of PbS (26) -RNase was isolated by rapid amplification of cDNA ends (RACE) technology from Chinese white pear (Pyrus bretschneideri) cultivar “Hongpisu.” The cDNA sequence for PbS (26) -RNase was deposited in GenBank under accession number EU081888. At the amino acid level, the PbS (26) -RNase displayed the highest similarity (96.9%) with PcSa-RNase of P. communis, and only seven amino acid differences were present in the two S-RNases. Phylogenetic analysis of rosaceous S-RNases indicated that the PbS (26) -RNase clustered with maloideous S-RNases, forming a subfamily-specific not a species-specific group. The PbS (26) -RNase gene was specifically expressed in the style but not other tissues/organs. The expression level of the PbS (26) -RNase gene rapidly increased at bell balloon stage (BBS), and then it dropped after pollination. However, the abundance of the PbS (26) -RNase gene transcript in the style was greater after cross-pollination than after self-pollination. In addition, a method for rapidly detecting the PbS (26) -RNase gene was developed via allele-specific primers design. The present study could provide a scientific basis for fully clarifying the mechanism of pear SI at the molecular level

Regulation of expression by promoters versus internal ribosome entry site in the 5′-untranslated sequence of the human cyclin-dependent kinase inhibitor p27

p27 kip1 regulates cell proliferation by binding to and inhibiting the activity of cyclin-dependent kinases and its expression oscillates with cell cycle. Recently, it has been suggested from studies using the traditional dicistronic DNA assay that the expression of p27 kip1 is regulated by internal ribosome entry site (IRES)-mediated translation initiation, and several RNA-binding protein factors were thought to play some role in this regulation. Considering the inevitable drawbacks of the dicistronic DNA assay, which could mislead a promoter activity or alternative splicing to IRES as previously demonstrated, we decided to reanalyze the 5′-untranslated region (5′-UTR) sequence of p27 kip1 and test whether it contains an IRES element or a promoter using more stringent methods, such as dicistronic RNA and promoterless dicistronic and monocistronic DNA assays. We found that the 5′-UTR sequence of human p27 kip1 does not have any significant IRES activity. The previously observed IRES activities are likely generated from the promoter activities present in the 5′-UTR sequences of p27 kip1 . The findings in this study indicate that transcriptional regulation likely plays an important role in p27 kip1 expression, and the mechanism of regulation of p27 expression by RNA-binding factors needs to be re-examined. The findings in this study also further enforce the importance that more stringent studies, such as promoterless dicistronic and monocistronic DNA and dicistronic RNA tests, are required to safeguard any future claims of cellular IRES

Regulation of Gene Expression by Internal Ribosome Entry Sites or Cryptic Promoters: the eIF4G Story

As an alternative to the scanning mechanism of initiation, the direct-internal-initiation mechanism postulates that the translational machinery assembles at the AUG start codon without traversing the entire 5′ untranslated region (5′-UTR) of the mRNA. Although the existence of internal ribosome entry sites (IRESs) in viral mRNAs is considered to be well established, the existence of IRESs in cellular mRNAs has recently been challenged, in part because when testing is carried out using a conventional dicistronic vector, Northern blot analyses might not be sensitive enough to detect low levels of monocistronic transcripts derived via a cryptic promoter or splice site. To address this concern, we created a new promoterless dicistronic vector to test the putative IRES derived from the 5′-UTR of an mRNA that encodes the translation initiation factor eIF4G. Our analysis of this 5′-UTR sequence unexpectedly revealed a strong promoter. The activity of the internal promoter relies on the integrity of a polypyrimidine tract (PPT) sequence that had been identified as an essential component of the IRES. The PPT sequence overlaps with a binding site for transcription factor C/EBPβ. Two other transcription factors, Sp1 and Ets, were also found to bind to and mediate expression from the promoter in the 5′-UTR of eIF4G mRNA. The biological significance of the internal promoter in the eIF4G mRNA might lie in the production of an N-terminally truncated form of the protein. Consistent with the idea that the cryptic promoter we identified underlies the previously reported IRES activity, we found no evidence of IRES function when a dicistronic mRNA containing the eIF4G sequence was translated in vitro or in vivo. Using the promoterless dicistronic vector, we also found promoter activities in the long 5′-UTRs of human Sno and mouse Bad mRNAs although monocistronic transcripts were not detectable on Northern blot analyses. The promoterless dicistronic vector might therefore prove useful in future studies to examine more rigorously the claim that there is IRES activity in cellular mRNAs

Parameter analysis of stability bearing capacity of bottom frame beam for container building

In this paper, the bottom frame beam of container building is analyzed by finite element method, and compared with the experimental results, the accuracy of the model is verified to meet the needs of the analysis. On this basis, by changing the stiffening position of the upper flange, the height of the web, the load layout and the thickness of the wall, the variation of the stability bearing capacity of the bottom frame beam was studied, the control factors affecting the stability bearing capacity of the bottom frame beam were obtained, and the selection suggestions were given

Refractory high-entropy alloys fabricated by powder metallurgy: Progress, challenges and opportunities

Refractory high-entropy alloys, which involve the mixing of four or more refractory metal elements in an equiatomic or near-equiatomic ratio, hold significant potential for various applications in high-temperature materials fields. This is mainly due to their stable phase structure and excellent high-temperature properties. While considerable interest has been in these alloys, most of them have been developed using melting casting technology. However, powder metallurgy has emerged as a promising alternative for further advancement in this field. It has the potential to expand the application areas and enhance the properties of these alloys. This article introduces to various techniques for fabricating pre-alloyed refractory high-entropy powders and their densification. Additionally, it reviews the methods for regulating the microstructure and properties of powder metallurgy refractory high-entropy alloys

Improving tensile strength of radial-additive friction stir repaired exceeded tolerance hole of 2024-T4 Al alloy by EHGWOA-BPNN

The radial-additive friction stir repairing (R-AFSR) process was used to successfully repair the exceeded tolerance hole of 2024-T4 aluminum alloy, and the formation and tensile strength of repaired joint were investigated. The enhanced hybridizing grey wolf optimization algorithm (EHGWOA) was innovatively proposed and had the advantages of strong search ability and large convergence speed. The weights and thresholds of back propagation neural network (BPNN) were optimized by the EHGWOA to enhance its prediction accuracy. Then, the EHGWOA-BPNN system was used to predict the joint strength and optimize the process parameters combination of R-AFSR process. The results showed that the repaired joint had not only the stir zone (SZ) with the thickness almost equal to the plate thickness but also no kissing bond defect in the SZ, thereby leading to the high tensile strength of the repaired joint. The maximum tensile strength of 308 MPa was obtained under the process parameters optimized by the EHGWOA-BPNN system, which was 4.5% higher than the maximum strength before optimization. The R-AFSR has great prospects to repair the exceeded tolerance holes of aluminum alloys, and the EHGWOA-BPNN system can be used to maximize the strength of repaired joint